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BACKGROUND Hematopoietic stem cells (HSC) have traditionally been frozen using the cryoprotectant DMSO in dextran-40, saline or albumin. However, the process of freezing and thawing results in loss of HSC numbers and/or function. METHODS This study investigated the use of CryoStor for the freezing of HSC from cord blood (CB). CB donations (n = 30) were collected under an Institutional Ethics Committee-approved protocol, volume reduced and frozen using three different methods of cryoprotection. Aliquots were frozen with either 10% DMSO in dextran-40, 10% DMSO in CryoStor or 5% DMSO in CryoStor. Prior to freezing samples were separated for nucleated cell (NC) and CD34+ counts and assessment of CD34+ viability. Aliquots were frozen and kept in vapor phase nitrogen for a minimum of 72 h. Vials were rapidly thawed at 37 degrees C and tested for NC and CD34+ counts and CD34+ viability and colony-forming unit (CFU) assay. RESULTS Cells frozen with CryoStor in 10% DMSO had significantly improved NC (P < 0.001), CD34+ recovery, viable CD34+ (P < 0.001) and CFU numbers (P < 0.001) compared with dextran in 10% DMSO. CryoStor in 5% DMSO resulted in significantly improved NC (P < 0.001) and CFU (P < 0.001). DISCUSSION These results suggest that improved HSC recovery, viability and functionality can be obtained using CryoStor with 10% DMSO and that similar if not better numbers can be obtained with 5% DMSO compared with dextran-40 with 10% DMSO.

Chronic treatment with antidepressants increases neurogenesis in the adult hippocampus. This increase in the production of new neurons may be required for the behavioral effects of antidepressants. However, it is not known which class of cells within the neuronal differentiation cascade is targeted by the drugs. We have generated a reporter mouse line, which allows identification and classification of early neuronal progenitors. It also allows accurate quantitation of changes induced by neurogenic agents in these distinct subclasses of neuronal precursors. We use this line to demonstrate that the selective serotonin reuptake inhibitor antidepressant fluoxetine does not affect division of stem-like cells in the dentate gyrus but increases symmetric divisions of an early progenitor cell class. We further demonstrate that these cells are the sole class of neuronal progenitors targeted by fluoxetine in the adult brain and suggest that the fluoxetine-induced increase in new neurons arises as a result of the expansion of this cell class. This finding defines a cellular target for antidepressant drug therapies.

Activation of thermogenic beige adipocytes has recently emerged as a promising therapeutic target in obesity and diabetes. Relevant human models for beige adipocyte differentiation are essential to implement such therapeutic strategies. We report a straightforward and efficient protocol to generate functional human beige adipocytes from human induced pluripotent stem cells (hiPSCs). Without overexpression of exogenous adipogenic genes, our method recapitulates an adipogenic developmental pathway through successive mesodermal and adipogenic progenitor stages. hiPSC-derived adipocytes are insulin sensitive and display beige-specific markers and functional properties, including upregulation of thermogenic genes, increased mitochondrial content, and increased oxygen consumption upon activation with cAMP analogs. Engraftment of hiPSC-derived adipocytes in mice produces well-organized and vascularized adipose tissue, capable of β-adrenergic-responsive glucose uptake. Our model of human beige adipocyte development provides a new and scalable tool for disease modeling and therapeutic screening.

We here report that doxycycline, an antibacterial agent, exerts dramatic effects on human embryonic stem and induced pluripotent stem cells (hESC/iPSCs) survival and self-renewal. The survival-promoting effect was also manifest in cultures of neural stem cells (NSCs) derived from hESC/iPSCs. These doxycycline effects are not associated with its antibacterial action, but mediated by direct activation of a PI3K-AKT intracellular signal. These findings indicate doxycycline as a useful supplement for stem cell cultures, facilitating their growth and maintenance. View Publication

Human adipose-derived stem cells (hASCs) become an appealing source for regenerative medicine. However, with the multi-passage or cryopreservation for large-scale growth procedures in terms of preclinical and clinical purposes, hASCs often reveal defective cell viability, which is a major obstacle for cell therapy. In our study, the effects of induced pluripotent stem cells-derived conditioned medium (iPS-CM) on the proliferation and anti-apoptosis in hASCs were investigated. hASCs at passage 1 were identified by the analysis of typical surface antigens with flow cytometry assay and adipogenic and osteogenic differentiation. The effect of iPS-CM on the proliferation in hASCs was analyzed by cell cycle assay and Ki67/P27 quantitative polymerase chain reaction analysis. The effect of iPS-CM on the anti-apoptosis of hASCs irradiated by 468 J/m(2) of ultraviolet C was investigated by annexin v/propidium iodide analysis, mitochondrial membrane potential assay, intracellular reactive oxygen species assay, Western blotting and caspase activity assays. The effect of iPS-CM on the surface antigen expressions of hASCs was analyzed using flow cytometry assay. The levels of Activin A and bFGF in culture supernatant of hASCs with different treatments were also detected by enzyme-linked immunosorbent assay. iPS-CM promoted proliferation and inhibited apoptosis of hASCs. This discovery demonstrates that iPS-CM might be used as one of the available means to overcome the propagation obstacle for hASCs and make for large-scale growth procedures in terms of preclinical and clinical purposes. View Publication

AIMS Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium, following acute myocardial infarction, may be, in part, due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials. MATERIALS & METHODS Bone marrow samples were taken from patients entered into the REGENERATE-IHD clinical trial after 5 days of subcutaneous granulocyte colony-stimulating factor injections. Each sample was divided equally for BM-MNC isolation using Ficoll-Paque and Lymphoprep, keeping all other procedural steps constant. Isolated fractions were characterized for hematopoietic stem cells, endothelial progenitor cells, T lymphocytes, B lymphocytes and NK cells using cell surface markers CD34(+), CD133(+)VEGFR2(+), CD45(+)CD3(+), CD45(+)CD19(+) and CD45(+)CD16(+)CD56(+), respectively. There were no significant differences in the absolute numbers and percentage cell recovery of various mononuclear cell types recovered following separation using either density gradient media. Cell viability and the proportion of various cell phenotypes investigated were similar between the two media. They were also equally efficient in excluding unwanted red blood cells, granulocytes and platelets from the final cell products. CONCLUSION We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep.

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript, we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore, we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages, further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication

Granulocyte colony-stimulating factor (G-CSF), the prototypical mobilizing cytokine, induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated, in part, through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization, osteoblast suppression, and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact, demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover, G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally, we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together, these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance, ultimately leading to HSPC mobilization.

Up to now, no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes, which hampers its application in gene therapy and immunotherapy areas. Here, we report that LVs incorporating measles virus (MV) glycoproteins, H and F, on their surface allowed transduction of 50&percnt; of quiescent B cells, which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation. Moreover, the naive and memory phenotypes of transduced resting B cells were maintained. Importantly, H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells, B-cell chronic lymphocytic leukemia cells, blocked in G(0)/G(1) early phase of the cell cycle. Thus, H/F-LV transduction overcomes the limitations of current LVs by making B cell-based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in these healthy and cancer immune cells.

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency, and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3, H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers, such as OCT4 (POU5F1), TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors.

PURPOSE: Preclinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However, one of the challenges in screening anticancer therapies is the assessment of off-target human toxicity. There is a need for in vivo models that can simulate efficacy and toxicities of promising therapeutic regimens. For example, hematopoietic cells of human origin are particularly sensitive to a variety of chemotherapeutic regimens, but in vivo models to assess potential toxicities have not been developed. In this study, a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity. EXPERIMENTAL DESIGN: A proof-of-concept, temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing because it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD.Cg-Prkdc(scid)IL2rg(tm1Wjl)/Sz (NOD/SCID/γchain(null)), reconstituted with human hematopoietic cells, and the impact of treatment on human hematopoiesis was evaluated. RESULTS: The dose-intensive regimen resulted in significant decreases in growth of human glioblastoma xenografts. When this regimen was administered to mice containing humanized bone marrow, flow cytometric analyses indicated that the human bone marrow cells were significantly more sensitive to treatment than the murine bone marrow cells and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (progenitor, lymphoid, and myeloid). CONCLUSIONS: The humanized bone marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anticancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation.

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94&percnt; of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90&percnt; reduced) and insulin-positive (65&percnt; reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult &dollar;&dollar;-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.