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Mesenchymal stromal progenitor cells (MSCs) are multipotent progenitors that can be isolated from numerous tissues. MSCs can undergo osteogenic differentiation under proper stimuli. We have recently demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most osteogenic BMPs. As one of the least studied BMPs, BMP9 has been shown to regulate angiogenesis in endothelial cells. However, it is unclear whether BMP9-regulated angiogenic signaling plays any important role in the BMP9-initiated osteogenic pathway in MSCs. Here, we investigate the functional role of hypoxia-inducible factor 1α (HIF1α)-mediated angiogenic signaling in BMP9-regulated osteogenic differentiation of MSCs. We find that BMP9 induces HIF1α expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1α potentiates BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo. siRNA-mediated silencing of HIF1α or HIF1α inhibitor CAY10585 profoundly blunts BMP9-induced osteogenic signaling in MSCs. HIF1α expression regulated by cobalt-induced hypoxia also recapitulates the synergistic effect between HIF1α and BMP9 in osteogenic differentiation. Mechanistically, HIF1α is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling and osteogenic signaling in MSCs. Thus, our findings should not only expand our understanding of the molecular basis behind BMP9-regulated osteoblastic lineage-specific differentiation, but also provide an opportunity to harness the BMP9-induced synergy between osteogenic and angiogenic signaling pathways in regenerative medicine.

In a previous study of prevalidation, a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed, to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up, by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper, we describe an international blind trial designed to apply this model to the clinical neutropenia, by testing 20 drugs, including 14 antineoplastics (Cytosar-U, 5-Fluorouracil, Myleran, Thioguanine, Fludarabine, Bleomycin, Methotrexate, Gemcitabine, Carmustine, Etoposide, Teniposide, Cytoxan, Taxol, Adriamycin); two antivirals (Retrovir, Zovirax,); three drugs for other therapeutic indications (Cyclosporin, Thorazine, Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin, Bleomycin, Etoposide, Fludarabine, 5-Fluorouracil, Myleran, Taxol, Teniposide, Thioguanine, and Thorazine), IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine, Cyclosporin, Cytosar-U, Cytoxan, Gemcitabine, Indocin, Lindane, Methotrexate, Retrovir, and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD�

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD, induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs, NPCs, and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition, GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons, but intriguingly, those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes, sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings, providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge, this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease.

Autologous feeder cells have been developed by various methods to minimize the presence of xenogenic entities in human embryonic stem cell (hESC) cultures. However, there was no systematic comparison of supportive effects of the feeder cells on hESC growth, nor comparison to the supportive effects of various feeder-free culture systems and standard mouse feeder cells. In this study, we aimed to compare the supportive abilities of autologous feeders derived either directly from H9 hESCs (H9 dF) or from outgrowth of embryoid body predifferentiated in suspension from H9 hESCs (H9 ebF). Mouse feeder system and matrigel-mTeSR1 feeder-free system were used as controls. H9 ebF was found to secrete more basic fibroblast growth factor in the conditioned medium than H9 dF did. The undifferentiated state of H9 hESCs was sustained more stably on H9 ebF than on H9 dF, and the differentiation potential of H9 hESCs on H9 ebF was higher than on H9 dF. We concluded that H9 ebF was an optimal autologous feeder to maintain the long-term undifferentiated state of hESCs in our current culture system. This study helps to standardize the autologous culture of hESCs. It also suggests a more definite direction for future development of xeno-free culture system for hESCs.

The development of the mature epidermis requires a coordinated sequence of signaling events and transcriptional changes to specify surface ectodermal progenitor cells to the keratinocyte lineage. The initial events that specify epidermal keratinocytes from ectodermal progenitor cells are not well understood. Here, we use both developing mouse embryos and human embryonic stem cells (hESCs) to explore the mechanisms that direct keratinocyte fate from ectodermal progenitor cells. We show that both hESCs and murine embryos express p63 before keratin 14. Furthermore, we find that Notch signaling is activated before p63 expression in ectodermal progenitor cells. Inhibition of Notch signaling pharmacologically or genetically reveals a negative regulatory role for Notch signaling in p63 expression during ectodermal specification in hESCs or mouse embryos, respectively. Taken together, these data reveal a role for Notch signaling in the molecular control of ectodermal progenitor cell specification to the epidermal keratinocyte lineage. View Publication

The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell, we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was as follows: hASC-iPSC, human embryonic stem cell, fibroblast-iPSC, fibroblasts, and, lastly, as the stiffest cell, hASC. In comparing hASC-iPSCs to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence, material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state.

Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High-fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 $$ textperiodcentered cm(2) on day 3 on chip and were sustained above 2000 $$ textperiodcentered cm(2) up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response. Biotechnol. Bioeng. 2016;9999: 1-11. textcopyright 2016 Wiley Periodicals, Inc.

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment. View Publication

Pluripotent stem cell (PSC) usage in heart regenerative medicine requires producing enriched cardiomyocytes (CMs) with mature phenotypes in a defined medium. However, current methods are typically performed in 2D environments that produce immature CMs. Here we report a simple, growth factor-free 3D culture system to rapidly and efficiently generate 85.07 ± 1.8% of spontaneously contractile cardiac spheres (scCDSs) using 3D-cultured human and monkey PSC-spheres. Along with small molecule-based 3D induction, this protocol produces CDSs of up to 95.7% CMs at a yield of up to 237 CMs for every input pluripotent cell, is effective for human and monkey PSCs, and maintains 81.03 ± 12.43% of CDSs in spontaneous contractibility for over three months. These CDSs displayed CM ultrastructure, calcium transient, appropriate pharmacological responses and CM gene expression profiles specific for maturity. Furthermore, 3D-derived CMs displayed more mature phenotypes than those from a parallel 2D-culture. The system is compatible to large-scaly produce CMs for disease study, cell therapy and pharmaceutics screening.

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.

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

Generating robust CD4+ T-helper cell type 1 (Th1) responses is essential for protective vaccine-induced type 1 immunity. Here, we examine whether immunization formulation associated with enhanced vaccine efficacy promotes antigen targeting and cell recruitment into lymph node (LN) niches associated with optimal type 1 responses. Immunization with antigen and Toll-like receptor agonist emulsified in oil leads to an increased differentiation of IFN$\gamma$/TNF-$\alpha$+ polyfunctional Th1 cells compared to an identical immunization in saline. Oil immunization results in a rapid delivery and persistence of antigen in interfollicular regions (IFRs) of the LN, whereas without oil, antigen is distributed in the medullary region. Following oil immunization, CXCL10-producing inflammatory monocytes accumulate in the IFR, which mobilizes antigen-specific CD4+ T cells into this niche. In this microenvironment, CD4+ T cells are advantageously positioned to encounter arriving IL-12-producing inflammatory dendritic cells (DCs). These data suggest that formulations delivering antigen to the LN IFR create an inflammatory niche that can improve vaccine efficacy.