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Human induced pluripotent stem cells (iPSC) have the potential to radically reduce the number of animals used in both toxicological science and disease elucidation. One initial obstacle culturing iPSC is that they require daily medium exchange. This study attempts to clarify why and propose some practical solutions. Two iPSC lineages were fed at different intervals in a full growth area (FGA) or a restricted growth area (RGA). The FGA consisted of a well coated with Matrigel™ and the RGA consisted of a coated coverslip placed in a well. Glucose,lactate,extracellular pH and cell cycle phases were quantified. Without daily feeding,FGA cultured iPSC had significantly reduced growth rates by day 2 and began to die by day 3. In contrast,RGA cultured cells grew to confluence over 3 days. Surprisingly,glucose was not exhausted under any condition. However,extracellular pH reached 6.8 after 72 h in FGA cultures. Artificially reducing medium pH to 6.8 also inhibited glycolysis and initiated an increase in G0/G1 phase of the cell cycle,while adding an additional 10 mM bicarbonate to the medium increased glycolysis rates. This study demonstrates that iPSC are highly sensitive to extracellular acidification,a likely limiting factor in maintenance of proliferative and pluripotent status. Culturing iPSC in RGA prevents rapid extracellular acidification,while still maintaining pluripotency and allowing longer feeding cycles.

Monocytes are key cells of the innate immune system. Their phenotypic and functional roles have been investigated in humans,mice and other animals,such as the rat,pig and cow. To date,detailed phenotypic analysis of monocytes has not been undertaken in dogs. Two important surface markers in human monocytes are CD14 and MHC class II (MHC II). By flow cytometry,we demonstrated that canine monocytes can be subdivided into three separate populations: CD14posMHC IIneg,CD14posMHC IIpos and CD14negMHC IIpos. Both light and transmission electron microscopy confirmed the monocytic identity of all three populations. The CD14posMHC IIneg population could be distinguished on an ultrastructural level by their smaller size,the presence of more numerous,larger granules,and more pseudopodia than both of the other populations. View Publication

Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons,animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus,our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons,extremely low heterogeneity of STLV sequences within each baboon,no evidence for superinfection within each baboon,and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed,premature stop codons were observed in 7{\%} and 56{\%} of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752,respectively.IMPORTANCE It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventive vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons,the identity of the viral gene product that is the major target of cellular immune responses,the persistence of viral amino acid sequences that are the major targets of cellular immune responses,and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what are seen with HTLV infection of humans. These results justify the use of STLV-infected baboons as a model system for vaccine development efforts. View Publication

Recently,many hurdles and limitations for production of clinically applicable iPSC derivatives have been overcome. Transgene-free iPSCs can be efficiently derived from easily accessible cell sources such as blood. Here we describe the generation of transgene-free hiPS cells from cord blood derived CD34+ cells,reprogrammed using CytoTune™ Sendai reprogramming vectors. CD34+ cell isolation,cultivation,reprogramming and establishment of resulting hiPSC lines were performed under the exclusive usage of animal-derived component-free (ADCF) materials and components. View Publication

A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article,we show that EPCR-positive UM171-treated cells,as opposed to EPCR-negative cells,exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers,such as CD38,EPCR expression is maintained when cells are introduced in culture,irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity,its expression is required for the repopulating activity of human HSCs. Altogether,our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs. View Publication

With increasing worldwide demand for safe blood,there is much interest in generating red blood cells in vitro as an alternative clinical product. However,available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply,and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach,immortalizing early adult erythroblasts generating a stable line,which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature,functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level,and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture. View Publication

Background: Local ischemia is the main pathological performance in osteonecrosis of the femoral head (ONFH). There is currently no effective therapy to promote angiogenesis in the femoral head. Recent studies revealed that exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSC-Exos) have great therapeutic potential in ischemic tissues,but whether they could promote angiogenesis in ONFH has not been reported,and little is known regarding the underlying mechanism. Methods: iPS-MSC-Exos were intravenously injected to a steroid-induced rat osteonecrosis model. Samples of the femoral head were obtained 3 weeks after all the injections. The effects were assessed by measuring local angiogenesis and bone loss through histological and immunohistochemical (IHC) staining,micro-CT and three-dimensional microangiography. The effects of exosomes on endothelial cells were studied through evaluations of proliferation,migration and tube-forming analyses. The expression levels of angiogenic related PI3K/Akt signaling pathway of endothelial cells were evaluated following stimulation of iPS-MSC-Exos. The promoting effects of exosomes were re-evaluated following blockade of PI3K/Akt. Results: The in vivo study revealed that administration of iPS-MSC-Exos significantly prevented bone loss,and increased microvessel density in the femoral head compared with control group. We found that iPS-MSC-Exos significantly enhanced the proliferation,migration and tube-forming capacities of endothelial cells in vitro. iPS-MSC-Exos could activate PI3K/Akt signaling pathway in endothelial cells. Moreover,the promoting effects of iPS-MSC-Exos were abolished after blockade of PI3K/Akt on endothelial cells. Conclusions: Our findings suggest that transplantation of iPS-MSC-Exos exerts a preventative effect on ONFH by promoting local angiogenesis and preventing bone loss. The promoting effect might be attributed to activation of the PI3K/Akt signaling pathway on endothelial cells. The data provide the first evidence for the potential of iPS-MSC-Exos in treating ONFH. View Publication

Retinal degenerative diseases are among the leading causes of blindness worldwide,and cell replacement is considered as a promising therapeutic. However,the resources of seed cells are scarce. To further explore this type of therapy,we adopted a culture system that could harvest a substantial quantity of retinal progenitor cells (RPCs) from human embryonic stem cells (hESCs) within a relatively short period of time. Furthermore,we transplanted these RPCs into the subretinal spaces of Royal College of Surgeons (RCS) rats. We quantified the thickness of the treated rats' outer nuclear layers (ONLs) and explored the visual function via electroretinography (ERG). It was found that the differentiated cells expressed RPC markers and photoreceptor progenitor markers. The transplanted RPCs survived for at least 12 weeks,resulting in beneficial effects on the morphology of the host retina,and led to a significant improvement in the visual function of the treated animals. These therapeutic effects suggest that the hESCs-derived RPCs could delay degeneration of the retina and partially restore visual function. View Publication

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration,but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs),endothelial cells (hiPSC-ECs),and smooth muscle cells (SMCs) are often limited by low yield,purity,and/or poor phenotypic stability. Here,we present novel protocols for generating hiPSC-CMs,-ECs,and -SMCs that are substantially more efficient than conventional methods,as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells,by sealing the needle track to prevent the cells from being squeezed out of the myocardium,and cell survival,by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury,the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch,and treatment with both the cells and the patch,but not with the cells alone,was associated with significant improvements in cardiac function and infarct size. View Publication

Somites form during embryonic development and give rise to unique cell and tissue types,such as skeletal muscles and bones and cartilage of the vertebrae. Using somitogenesis-stage human embryos,we performed transcriptomic profiling of human presomitic mesoderm as well as nascent and developed somites. In addition to conserved pathways such as WNT-$\beta$-catenin,we also identified BMP and transforming growth factor $\beta$ (TGF-$\beta$) signaling as major regulators unique to human somitogenesis. This information enabled us to develop an efficient protocol to derive somite cells in vitro from human pluripotent stem cells (hPSCs). Importantly,the in-vitro-differentiating cells progressively expressed markers of the distinct developmental stages that are known to occur during in vivo somitogenesis. Furthermore,when subjected to lineage-specific differentiation conditions,the hPSC-derived somite cells were multipotent in generating somite derivatives,including skeletal myocytes,osteocytes,and chondrocytes. This work improves our understanding of human somitogenesis and may enhance our ability to treat diseases affecting somite derivatives. View Publication

Temporal manipulation of the in vitro environment and growth factors can direct differentiation of human pluripotent stem cells into organoids - aggregates with multiple tissue-specific cell types and three-dimensional structure mimicking native organs. A mechanistic understanding of early organoid formation is essential for improving the robustness of these methods,which is necessary prior to use in drug development and regenerative medicine. We investigated intestinal organoid emergence,focusing on measurable parameters of hindgut spheroids,the intermediate step between definitive endoderm and mature organoids. We found that 13{\%} of spheroids were pre-organoids that matured into intestinal organoids. Spheroids varied by several structural parameters: cell number,diameter and morphology. Hypothesizing that diameter and the morphological feature of an inner mass were key parameters for spheroid maturation,we sorted spheroids using an automated micropipette aspiration and release system and monitored the cultures for organoid formation. We discovered that populations of spheroids with a diameter greater than 75 $\mu$m and an inner mass are enriched 1.5- and 3.8-fold for pre-organoids,respectively,thus providing rational guidelines towards establishing a robust protocol for high quality intestinal organoids. View Publication

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach,often involving the use of viral vectors. Electroporation using square-wave generating devices,like Lonza's Nucleofector,is a widely used option,but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work,we show that our in-house developed buffers,termed Chicabuffers,can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device,we electroporated 14 different cell lines and also primary cells,like mesenchymal stem cells and cord blood CD34+,providing optimized protocols for each of them. Moreover,when combined with sleeping beauty-based transposon system,long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells,facilitating the widespread adoption of this technology. View Publication