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Kelly et al . describe antibodies for purifying two progenitor populations that arise during the differentiation of human embryonic stem cells to pancreatic beta cells: pancreatic endoderm and polyhormonal endocrine cells. After transplantation into mice, only pancreatic endoderm cells differentiate into glucose-responsive insulin-producing 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.

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Purpose Calcineurin inhibitor use after allogeneic hematopoietic cell transplantation (allo-HCT) is associated with significant magnesium wasting. Utilization of a prolonged magnesium infusion is thought to lead to a lower serum peak concentration and therefore, decreased renal wasting of magnesium. In November 2017, our institution implemented a modification to our inpatient electrolyte replacement protocol for allo-HCT recipients that extended the magnesium infusion rate from 4 g/2 h to 4 g/4 h based on this theoretical advantage. The primary objective of this study was to compare the median magnesium requirements per day of admission between patients who received magnesium 4 g/2 h to patients who received magnesium 4 g/4 h. Secondary objectives included a comparison of the per-patient median serum magnesium concentration during admission, as well as the median incremental difference in serum magnesium concentration after intravenous replacement per patient per admission. Methods Allo-HCT recipients who received prolonged infusion magnesium infusions were compared to a historical cohort of allo-HCT patients who received shorter IV magnesium infusions. Admissions were included if the patient had received an allo-HCT within 100 days prior, was admitted to the Transplant and Cellular Therapy Unit at WVU Medicine J.W. Ruby Memorial Hospital, and received at least one magnesium infusion and one dose of cyclosporine or tacrolimus. Admissions were excluded if the patient received oral magnesium, total parenteral nutrition, aminoglycosides, amphotericin, carboplatin, cisplatin, or foscarnet. Results The pre-implementation group consisted of 81 admissions ( n  = 64 patients), while the post-implementation group consisted of 90 admissions ( n  = 60 patients). Median magnesium requirements per day of admission were not different between groups at 1.4 g of magnesium in the pre-implementation group and 1.9 g of magnesium in the post-implementation group ( P  = 0.25). Median serum magnesium concentrations and median incremental difference in serum magnesium concentration after intravenous replacement were also not different between groups: 1.65 mg/dL vs 1.60 mg/dL ( P  = 0.65) and 0.30 mg/dL vs 0.28 mg/dL ( P  = 0.67), respectively. Conclusions Prolonged infusion of magnesium in allo-HCT recipients receiving CNI therapy does not result in improvement in magnesium retention.

A theoretical pharmacokinetic interaction mediated through l-amino acid transporter 1 and 2 exists between gabapentin (GP) and pregabalin (PG) with melphalan. Peripheral neuropathy is a common toxicity of various multiple myeloma regimens commonly utilized prior to autologous hematopoietic cell transplant (auto-HCT) with high-dose melphalan (HD-Mel). Therefore, it is likely concurrent administration of either GP or PG will occur in patients receiving HD-Mel conditioning for auto-HCT, which could potentially increase cellular uptake and worsen the mucosal injury. A retrospective chart review of adult patients from January 2012 to July 2016 who received HD-Mel (140–200 mg/m2) at West Virginia University Medicine was performed to assess toxicity and outcomes in these patients. A total of 80 patients were included in the study, with 30 patients receiving GP or PG and 50 control patients. There were no significant differences in grade 2 or higher mucositis, admissions for nausea/vomiting/diarrhea, intravenous opioid requirements, oral topical therapies, antidiarrheal medication use, rescue anti-emetics, days of nausea or vomiting, pain scores, neutrophil or platelet engraftment, treatment-related mortality, progression-free survival, or overall survival. Our data suggest that it is safe to continue GP/PG therapy throughout HD-Mel therapy, with no negative transplant outcomes. Prospective studies or evaluations of larger databases are necessary to better characterize the clinical effect of concomitant therapy.

POU4 homologs are involved in the development of sensory cell types across diverse species, including cnidarians, ascidians, and mammals. Whether these developmental regulators are redeployed during adult tissue maintenance and regeneration remains an open question in regenerative biology. Here, we investigated the role of the Schmidtea mediterranea BRN3/POU4 homolog, Smed-pou4-2 ( pou4-2 ), in the regeneration of mechanosensory neurons. We found that pou4-2 is regulated by the SoxB1 homolog soxB1-2 and is expressed in a distinct population of ciliated sensory cells that detect water flow. Transcriptomic analysis of pou4-2 -deficient planarians revealed enrichment for conserved genes associated with human auditory and vestibular function, suggesting that planarian rheosensory neurons share molecular features with mammalian inner ear hair cells. Expression of these conserved genes was significantly reduced following RNAi-mediated knockdown of pou4-2 . To determine whether these transcriptional changes had functional consequences, we assessed the impact of pou4-2 knockdown on sensory function. pou4-2 RNAi resulted in impaired mechanosensation in both uninjured and regenerating planarians. Together with the loss of terminal differentiation markers in mechanosensory neurons, these findings identify Smed-pou4-2 as a key regulator of mechanosensory neuron identity in planarians and support the idea that conserved sensory specification programs are redeployed during adult tissue regeneration.

Background Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians. Results We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments. Conclusions The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand planarian biology and to characterize phenotypes following RNAi experiments. In addition, we present alterations to fixation protocols and demonstrate how these changes can increase the labeling efficiencies of antibodies used to stain whole planarians.

Background Planarians are renowned for their regenerative capacity and are an attractive model for the study of adult stem cells and tissue regeneration. In an effort to better understand the molecular mechanisms underlying planarian regeneration, we performed a functional genomics screen aimed at identifying genes involved in this process in Schmidtea mediterranea . Methods We used microarrays to detect changes in gene expression in regenerating and non-regenerating tissues in planarians regenerating one side of the head and followed this with high-throughput screening by in situ hybridization and RNAi to characterize the expression patterns and function of the differentially expressed genes. Results Along with five previously characterized genes ( Smed-cycD , Smed-morf41/mrg-1 , Smed-pdss2/dlp1, Smed-slbp , and Smed-tph ), we identified 20 additional genes necessary for stem cell maintenance ( Smed-sart3 , Smed-smarcc-1 , Smed-espl1 , Smed-rrm2b-1 , Smed-rrm2b-2 , Smed-dkc1 , Smed-emg1 , Smed-lig1 , Smed-prim2 , Smed-mcm7 , and a novel sequence) or general regenerative capability ( Smed-rbap46/48-2 , Smed-mcm2 , Smed-ptbp1 , and Smed-fen-1 ) or that caused tissue-specific defects upon knockdown ( Smed-ddc , Smed-gas8 , Smed-pgbd4 , and Smed-b9d2 ). We also found that a homolog of the nuclear transport factor Importin-α plays a role in stem cell function and tissue patterning, suggesting that controlled nuclear import of proteins is important for regeneration. Conclusions Through this work, we described the roles of several previously uncharacterized genes in planarian regeneration and implicated nuclear import in this process. We have additionally created an online database to house our in situ and RNAi data to make it accessible to the planarian research community.

Planarians are renowned for their regenerative capacity and are an attractive model for the study of adult stem cells and tissue regeneration. In an effort to better understand the molecular mechanisms underlying planarian regeneration, we performed a functional genomics screen aimed at identifying genes involved in this process in Schmidtea mediterranea. We used microarrays to detect changes in gene expression in regenerating and non-regenerating tissues in planarians regenerating one side of the head and followed this with high-throughput screening by in situ hybridization and RNAi to characterize the expression patterns and function of the differentially expressed genes. Along with five previously characterized genes (Smed-cycD, Smed-morf41/mrg-1, Smed-pdss2/dlp1, Smed-slbp, and Smed-tph), we identified 20 additional genes necessary for stem cell maintenance (Smed-sart3, Smed-smarcc-1, Smed-espl1, Smed-rrm2b-1, Smed-rrm2b-2, Smed-dkc1, Smed-emg1, Smed-lig1, Smed-prim2, Smed-mcm7, and a novel sequence) or general regenerative capability (Smed-rbap46/48-2, Smed-mcm2, Smed-ptbp1, and Smed-fen-1) or that caused tissue-specific defects upon knockdown (Smed-ddc, Smed-gas8, Smed-pgbd4, and Smed-b9d2). We also found that a homolog of the nuclear transport factor Importin-[alpha] plays a role in stem cell function and tissue patterning, suggesting that controlled nuclear import of proteins is important for regeneration. Through this work, we described the roles of several previously uncharacterized genes in planarian regeneration and implicated nuclear import in this process. We have additionally created an online database to house our in situ and RNAi data to make it accessible to the planarian research community.

Understanding how adult stem cells generate neurons is critical for advancing regenerative medicine. However, few in vivo models enable studying how stem cell fates are specified as neurons in an adult body. The planarian provides a powerful system for investigating these mechanisms, owing to its abundant adult pluripotent stem cells, termed neoblasts, and its capacity to regenerate a molecularly complex nervous system. The SoxB1 family of transcription factors is broadly implicated in ectodermal lineage commitment. In planarians, the SoxB1 homolog has been shown to promote neural and epidermal differentiation. However, the mechanisms by which influences chromatin dynamics and transcriptional programs during adult neurogenesis remain unknown. To address this, we performed ATAC-seq and RNA-seq on neural-rich head tissues to assess how RNAi knockdown alters chromatin accessibility and gene expression. Disrupting resulted in reduced chromatin accessibility and transcriptional downregulation at neural and epidermal loci, consistent with a pioneer-like role in chromatin priming. We identified 31 candidate downstream targets with concordant accessibility and expression changes, including the transcription factors and , which regulate mechanosensory and ion transport genes. Head tissue sampling enabled the detection of -responsive genes within rare neural subtypes that were missed in our previous whole-body RNA-seq experiments. These findings offer mechanistic insight into adult ectodermal lineage specification and establish a framework for understanding chromatin-mediated neurogenesis in regenerative systems.