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BACKGROUND Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia, although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. METHODS We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling, insulin regulation of the major AD proteins Abeta$ and/or tau, and/or calcium fluxes is altered by the PSEN2 N141I mutation. RESULTS We report herein that wildtype, PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Abeta$42/40 ratio. Unexpectedly, we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux, which could be prevented by chronic exposure of BFCNs to insulin. CONCLUSIONS Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Abeta$42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence, our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD.

Although CD31 expression on human thymocytes has been reported, a detailed analysis of CD31 expression at various stages of T cell development in the human thymus is missing. In this study, we provide a global picture of the evolution of CD31 expression from the CD34(+) hematopoietic precursor to the CD45RA(+) mature CD4(+) and CD8(+) single-positive (SP) T cells. Using nine-color flow cytometry, we show that CD31 is highly expressed on CD34(+) progenitors and stays high until the early double-positive stage (CD3(-)CD4(+)CD8α(+)β(-)). After β-selection, CD31 expression levels become low to undetectable. CD31 expression then increases and peaks on CD3(high)CD4(+)CD8(+) double-positive thymocytes. However, following positive selection, CD31 expression differs dramatically between CD4(+) and CD8(+) lineages: homogeneously high on CD8 SP but lower or negative on CD4 SP cells, including a subset of CD45RA(+)CD31(-) mature CD4(+) thymocytes. CD31 expression on TCRγδ thymocytes is very similar to that of CD4 SP cells. Remarkably, there is a substantial subset of semimature (CD45RA(-)) CD4 SP thymocytes that lack CD31 expression. Moreover, FOXP3(+) and ICOS(+) cells are overrepresented in this CD31(-) subpopulation. Despite this CD31(-)CD45RA(-) subpopulation, most egress-capable mature CD45RA(+) CD4 SP thymocytes express CD31. The variations in CD31 expression appear to coincide with three major selection processes occurring during thymopoiesis: β-selection, positive selection, and negative selection. Considering the ability of CD31 to modulate the TCR's activation threshold via the recruitment of tyrosine phosphatases, our results suggest a significant role for CD31 during T cell development.

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile.

Induced pluripotent stem cell (iPSC) therapeutics are a promising treatment for genetic and infectious diseases. To assess engraftment, risk of neoplastic formation, and therapeutic benefit in an autologous setting, testing iPSC therapeutics in an appropriate model, such as the pigtail macaque (Macaca nemestrina; Mn), is crucial. Here, we developed a chemically defined, scalable, and reproducible specification protocol with bone morphogenetic protein 4, prostaglandin-E2 (PGE2), and StemRegenin 1 (SR1) for hematopoietic differentiation of Mn iPSCs. Sequential coculture with bone morphogenetic protein 4, PGE2, and SR1 led to robust Mn iPSC hematopoietic progenitor cell formation. The combination of PGE2 and SR1 increased CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cell yield by 6-fold. CD34(+)CD38(-)Thy1(+)CD45RA(-)CD49f(+) cells isolated on the basis of CD34 expression and cultured in SR1 expanded 3-fold and maintained this long-term repopulating HSC phenotype. Purified CD34(high) cells exhibited 4-fold greater hematopoietic colony-forming potential compared with unsorted hematopoietic progenitors and had bilineage differentiation potential. On the basis of these studies, we calculated the cell yields that must be achieved at each stage to meet a threshold CD34(+) cell dose that is required for engraftment in the pigtail macaque. Our protocol will support scale-up and testing of iPSC-derived CD34(high) cell therapies in a clinically relevant nonhuman primate model.

BACKGROUND Individuals with 22q11.2 Deletion Syndrome (22q11.2 DS) are a specific high-risk group for developing schizophrenia (SZ), schizoaffective disorder (SAD) and autism spectrum disorders (ASD). Several genes in the deleted region have been implicated in the development of SZ, e.g., PRODH and DGCR8. However, the mechanistic connection between these genes and the neuropsychiatric phenotype remains unclear. To elucidate the molecular consequences of 22q11.2 deletion in early neural development, we carried out RNA-seq analysis to investigate gene expression in early differentiating human neurons derived from induced pluripotent stem cells (iPSCs) of 22q11.2 DS SZ and SAD patients. METHODS Eight cases (ten iPSC-neuron samples in total including duplicate clones) and seven controls (nine in total including duplicate clones) were subjected to RNA sequencing. Using a systems level analysis, differentially expressed genes/gene-modules and pathway of interests were identified. Lastly, we related our findings from in vitro neuronal cultures to brain development by mapping differentially expressed genes to BrainSpan transcriptomes. RESULTS We observed ˜2-fold reduction in expression of almost all genes in the 22q11.2 region in SZ (37 genes reached p-value textless 0.05, 36 of which reached a false discovery rate textless 0.05). Outside of the deleted region, 745 genes showed significant differences in expression between SZ and control neurons (p textless 0.05). Function enrichment and network analysis of the differentially expressed genes uncovered converging evidence on abnormal expression in key functional pathways, such as apoptosis, cell cycle and survival, and MAPK signaling in the SZ and SAD samples. By leveraging transcriptome profiles of normal human brain tissues across human development into adulthood, we showed that the differentially expressed genes converge on a sub-network mediated by CDC45 and the cell cycle, which would be disrupted by the 22q11.2 deletion during embryonic brain development, and another sub-network modulated by PRODH, which could contribute to disruption of brain function during adolescence. CONCLUSIONS This study has provided evidence for disruption of potential molecular events in SZ patient with 22q11.2 deletion and related our findings from in vitro neuronal cultures to functional perturbations that can occur during brain development in SZ.

AIM To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics, colony-forming potential, as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS hESC-MSC showed similar morphology, and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications. View Publication

BACKGROUND Patient-derived tumor models are the new standard for pre-clinical drug testing and biomarker discovery. However, the emerging technology of primary pancreatic cancer organoids has not yet been broadly implemented in research, and complex organotypic models using organoids in co-culture with stromal and immune cellular components of the tumor have yet to be established. In this study, our objective was to develop and characterize pancreatic cancer organoids and multi-cell type organotypic co-culture models to demonstrate their applicability to the study of pancreatic cancer. METHODS We employed organoid culture methods and flow cytometric, cytologic, immunofluorescent and immunohistochemical methods to develop and characterize patient-derived pancreatic cancer organoids and multi-cell type organotypic co-culture models of the tumor microenvironment. RESULTS We describe the culture and characterization of human pancreatic cancer organoids from resection, ascites and rapid autopsy sources and the derivation of adherent tumor cell monocultures and tumor-associated fibroblasts from these sources. Primary human organoids displayed tumor-like cellular morphology, tissue architecture and polarity in contrast to cell line spheroids, which formed homogenous, non-lumen forming spheres. Importantly, we demonstrate the construction of complex organotypic models of tumor, stromal and immune components of the tumor microenvironment. Activation of myofibroblast-like cancer associated fibroblasts and tumor-dependent lymphocyte infiltration were observed in these models. CONCLUSIONS These studies provide the first report of novel and disease-relevant 3D in-vitro models representing pancreatic tumor, stromal and immune components using primary organoid co-cultures representative of the tumor-microenvironment. These models promise to facilitate the study of tumor-stroma and tumor-immune interaction and may be valuable for the assessment of immunotherapeutics such as checkpoint inhibitors in the context of T-cell infiltration.

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets, which can account for c-myb knockdown effects. Indeed, chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4high cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation, efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved, while retaining their pluripotency. When added during the reprogramming process, CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus, CD30-LV may serve as novel tool for the selective gene transfer into pluripotent stem cells with broad applications in basic and therapeutic research. View Publication

Although regenerative medicine products are at the forefront of scientific research, technological innovation, and clinical translation, their reproducibility and large-scale production are compromised by automation, monitoring, and standardization issues. To overcome these limitations, new technologies at software (e.g., algorithms and artificial intelligence models, combined with imaging software and machine learning techniques) and hardware (e.g., automated liquid handling, automated cell expansion bioreactor systems, automated colony-forming unit counting and characterization units, and scalable cell culture plates) level are under intense investigation. Automation, monitoring and standardization should be considered at the early stages of the developmental cycle of cell products to deliver more robust and effective therapies and treatment plans to the bedside, reducing healthcare expenditure and improving services and patient care.

Avian species are important model animals for developmental biology and disease research. However, unlike in mice, where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function, clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs), the first nonmammalian iPSCs, which were clonally isolated and propagated, important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes, indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes, oligodendrocytes, and neurons. Ultimately, qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers, extraembryonic tissues, and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals.

How BMP signaling integrates into and destabilizes the pluripotency circuitry of human pluripotent stem cells (hPSCs) to initiate differentiation into individual germ layers is a long-standing puzzle. Here we report muscle segment homeobox 2 (MSX2), a homeobox transcription factor of msh family, as a direct target gene of BMP signaling and a master mediator of hPSCs' differentiation to mesendoderm. Enforced expression of MSX2 suffices to abolish pluripotency and induce directed mesendoderm differentiation of hPSCs, while MSX2 depletion impairs mesendoderm induction. MSX2 is a direct target gene of the BMP pathway in hPSCs, and can be synergistically activated by Wnt signals via LEF1 during mesendoderm induction. Furthermore, MSX2 destabilizes the pluripotency circuitry through direct binding to the SOX2 promoter and repression of SOX2 transcription, while MSX2 controls mesendoderm lineage commitment by simultaneous suppression of SOX2 and induction of NODAL expression through direct binding and activation of the Nodal promoter. Interestingly, SOX2 can promote the degradation of MSX2 protein, suggesting a mutual antagonism between the two lineage-specifying factors in the control of stem cell fate. Together, our findings reveal crucial new mechanisms of destabilizing pluripotency and directing lineage commitment in hPSCs.