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The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-,atrial-,and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives,which in turn promoted cardiomyocyte differentiation. Moreover,a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation,improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation,measuring action potentials,and intracellular Ca(2+) dynamics. These findings are relevant for improving our understanding on human heart development,and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes,similar to those observed in adult cardiac myocytes. View Publication

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus,new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors,and Triple-Negative" Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor� View Publication

The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however,the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here,we report the directed differentiation of CHX10(+) V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid,sonic hedgehog,and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10(+) cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time,CHX10(+) cells expressed neuronal markers [neurofilament,NeuN,and vesicular glutamate transporter 2 (VGlut2)],and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10(+) cells within the differentiated population,which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice,hPSC-derived V2a cultures survived at the site of injection,coexpressed NeuN and VGlut2,extended neurites textgreater5 mm,and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI. View Publication

The authors surveyed whole-exome and RNA-sequencing data from 252 unique pluripotent stem cell lines,some of which are in the pipeline for clinical use,and found that approximately 5{\%} of cell lines had acquired mutations in the TP53 gene that allow mutant cells to rapidly outcompete non-mutant cells,but do not prevent differentiation. View Publication

Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential,however,is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here,we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG,we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes,and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane,suggesting that the ectodomain is not required for protein loading. Finally,exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain,confirming a role of the ectodomain in cell tropism. In summary,our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells. View Publication

Recent studies using defined transcription factors to convert skin fibroblasts into chondrocytes have raised the question of whether osteo-chondroprogenitors expressing SOX9 and RUNX2 could also be generated during the course of the reprogramming process. Here,we demonstrated that doxycycline-inducible expression of reprogramming factors (KLF4 [K] and c-MYC [M]) for 6 days were sufficient to convert murine fibroblasts into SOX9(+)/RUNX2(+) cellular aggregates and together with SOX9 (S) promoted the conversion efficiency when cultured in a defined stem cell medium,mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in vitro,but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether,we provide a reprogramming strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues. View Publication

The proliferation and differentiation effects of the synthetic retinoid TTNPB and of 13-cis retinoic acid (RA) on hemopoietic progenitors from bone marrow of myelodysplastic syndrome (MDS) patients were compared. The addition of TTNPB or RA to culture plates containing MDS patient's marrow cells stimulated myeloid colony (CFU-C) growth and caused a significant increase in granulocytic colonies (CFU-G). In the presence of RA the increase in CFU-G was statistically insignificant. Cellular differentiation studies in liquid suspension culture revealed that the two retinoic acid analogues cause a marked decrease in immature granulocytes and an increase in mature granulocytes. There was further an increase in the number of cells that reacted positively with monoclonal antibodies (McAb) binding specifically to granulocytes (B4,3,B13,9 and Leu M4) and a decrease in the percentage of cells reacting with the McAb against Ia-like determinants. These findings indicate that TTNPB is as active as RA in stimulating the growth of hemopoietic progenitors from MDS patients and in enhancing granulocytic differentiation in liquid culture. View Publication

To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells,a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells,purified from umbilical cord blood by flow cytometry and cell sorting,were selected for this study because of their high content of clonogenic cells (34%),particularly multipotent progenitors (CFU-MIX,12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF,IL-6,IL-3,and erythropoietin (Epo); (3) MGF,IL-6,granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein (FP),macrophage colony-stimulating factor (M-CSF),and granulocyte-CSF (G-CSF); and (4) MGF,IL-6,FP,M-CSF,G-CSF,and Epo. Maximum numbers of erythroid progenitors (BFU-E,up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF,IL-6,IL-3,and Epo,whereas maximum levels of myeloid progenitors (CFU-C,up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF,IL-6,FP,M-CSF,and G-CSF. When MGF,IL-6,FP,M-CSF,G-CSF,and Epo were present,maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore,the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors,but rather had a permissive effect on the development of lineage-restricted clonogenic cells. View Publication

Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells,which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered,the rank order of potency is oleoyl-LPA textgreater arachidonoyl-LPA textgreater linolenoyl-LPA textgreater linoleoyl-LPA textgreater stearoyl-LPA = palmitoyl-LPA textgreater myristoyl-LPA. The shorter-chain species,lauroyl- and decanoyl-LPA,show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses textgreater 15 microM. Similarly,the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate,a putative messenger with some structural similarities to LPA,elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however,cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that,although many features of the LPA structure are important for optimal activity,the phosphate group is most critical,suggesting that this moiety is directly involved in receptor activation. View Publication

Cytosolic aldehyde dehydrogenase (ALDH),an enzyme responsible for oxidizing intracellular aldehydes,has an important role in ethanol,vitamin A,and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues,including bone marrow and intestine,appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However,although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH,isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde,dansyl aminoacetaldehyde (DAAA),could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover,DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore,marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations. View Publication

Under appropriate conditions in culture,embryonic stem cells will differentiate and form embryoid bodies that have been shown to contain cells of the hematopoietic,endothelial,muscle and neuronal lineages. Many aspects of the lineage-specific differentiation programs observed within the embryoid bodies reflect those found in the embryo,indicating that this model system provides access to early cell populations that develop in a normal fashion. Recent studies involving the differentiation of genetically altered embryonic stem cells highlight the potential of this in vitro differentiation system for defining the function of genes in early development. View Publication

A high proportion of the CD34+CD38- cells in normal human marrow are defined as long-term culture-initiating cells (LTC-IC) because they can proliferate and differentiate when co-cultured with cytokine-producing stromal feeder layers. In contrast,very few CD34+CD38- cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC),although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38- cells in the latter type of culture showed that Flt3-ligand (FL),Steel factor (SF),and interleukin (IL)-3 were both necessary and sufficient to obtain an approximately 30-fold amplification of the input LTC-IC population within 10 d. As single factors,only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly,a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of textgreater 500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38- cells. However,this required the presence of IL-6 and/or granulocyte/colony-stimulating factor and/or nerve growth factor beta in addition to FL,SF,and IL-3. Also,for this response,the most potent single-acting factor tested was IL-3,not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facilitate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols. View Publication