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Generating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However,the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study,we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly,the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore,the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo. View Publication

Synaptic plasticity is mediated by the dynamic localization of proteins to and from synapses. This is controlled,in part,through activity-induced palmitoylation of synaptic proteins. Here we report that the ability of the palmitoyl-acyl transferase,DHHC5,to palmitoylate substrates in an activity-dependent manner is dependent on changes in its subcellular localization. Under basal conditions,DHHC5 is bound to PSD-95 and Fyn kinase,and is stabilized at the synaptic membrane through Fyn-mediated phosphorylation of a tyrosine residue within the endocytic motif of DHHC5. In contrast,DHHC5's substrate,δ-catenin,is highly localized to dendritic shafts,resulting in the segregation of the enzyme/substrate pair. Neuronal activity disrupts DHHC5/PSD-95/Fyn kinase complexes,enhancing DHHC5 endocytosis,its translocation to dendritic shafts and its association with δ-catenin. Following DHHC5-mediated palmitoylation of δ-catenin,DHHC5 and δ-catenin are trafficked together back into spines where δ-catenin increases cadherin stabilization and recruitment of AMPA receptors to the synaptic membrane. View Publication

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus,they are increasingly employed as models for cancer and drug research,as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures,and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore,SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells,providing a pathway to enable our understanding of morphogenesis in 3D cultures. View Publication

Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT),underscoring the need to improve the graft-versus-leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest,but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs,but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement,while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally,adoptive transfers of activated-pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL. View Publication

Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs),but little is known about SSEA-5 expression in other primitive tissues (e.g.,human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors,cell lines,teratomas,and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors,such as embryonal carcinoma. In tumor cell lines,SSEA-5 was highly immunoreactive in Capan-1 cells,while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs,with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use. View Publication

Chronic granulomatous disease (CGD) is an inherited immunodeficiency,caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD),which is due to mutations in the CYBB (gp91phox) gene,a component of NADPH oxidase,accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function,we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both,BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion,we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general. View Publication

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here,we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains,both lacking canonical export signals,are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs-named 'cyclonals'-effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. View Publication

Significant efforts have been invested into the differentiation of stem cells into functional hepatocyte-like cells that can be used for cell therapy,disease modeling and drug screening. Most of these efforts have been concentrated on the use of growth factors to recapitulate developmental signals under in vitro conditions. Using small molecules instead of growth factors would provide an attractive alternative since small molecules are cell-permeable and cheaper than growth factors. We have developed a protocol for the differentiation of human embryonic stem cells into hepatocyte-like cells using a predominantly small molecule-based approach (SM-Hep). This 3 step differentiation strategy involves the use of optimized concentrations of LY294002 and bromo-indirubin-3'-oxime (BIO) for the generation of definitive endoderm; sodium butyrate and dimethyl sulfoxide (DMSO) for the generation of hepatoblasts and SB431542 for differentiation into hepatocyte-like cells. Activin A is the only growth factor required in this protocol. Our results showed that SM-Hep were morphologically and functionally similar or better compared to the hepatocytes derived from the growth-factor induced differentiation (GF-Hep) in terms of expression of hepatic markers,urea and albumin production and cytochrome P450 (CYP1A2 and CYP3A4) activities. Cell viability assays following treatment with paradigm hepatotoxicants Acetaminophen,Chlorpromazine,Diclofenac,Digoxin,Quinidine and Troglitazone showed that their sensitivity to these drugs was similar to human primary hepatocytes (PHHs). Using SM-Hep would result in 67% and 81% cost reduction compared to GF-Hep and PHHs respectively. Therefore,SM-Hep can serve as a robust and cost effective replacement for PHHs for drug screening and development. View Publication

Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely defined xeno-free systems for large-scale cell expansion and differentiation. Previously,we demonstrated that microcarriers (MCs) coated with mouse laminin-111 (LN111) and positively charged poly-l-lysine (PLL) critically enable the formation and evolution of cells/MC aggregates with high cell yields obtained under agitated conditions. In this article,we further improved the MC system into a defined xeno-free MC one in which the MCs are coated with recombinant human laminin-521 (LN521) alone without additional positive charge. The high binding affinity of the LN521 to cell integrins enables efficient initial HES-3 cell attachment (87%) and spreading (85%),which leads to generation of cells/MC aggregates (400 $\$ in size) and high cell yields (2.4-3.5×10(6) cells/mL) within 7 days in agitated plate and scalable spinner cultures. The universality of the system was demonstrated by propagation of an induced pluripotent cells line in this defined MC system. Long-term pluripotent (textgreater90% expression Tra-1-60) cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover,tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved. The new LN521-based MC system offers a defined,xeno-free,GMP-compatible,and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on MCs enabled a 34% savings in matrix and media costs over monolayer cultures to produce 10(8) cells. View Publication

Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently,chromatin has been shown to play a part in this process. To elucidate this relationship further,we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed,that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally,cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts,and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed,our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer. View Publication

Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source,but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis,FH-iPSC were transfected with a 31 kb plasmid (pEHZ-LDLR-LDLR) containing a wild-type LDLR (FH-iPSC-LDLR) controlled by 10 kb of upstream genomic DNA as well as Epstein-Barr sequences (EBNA1 and oriP) for episomal retention and replication. After six months of selective culture,pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells,which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis,FH-iPSC ± LDLR were differentiated into mesenchymal cells (MC),pretreated with excess free sterols,Lovastatin,or ethanol (control),and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response,with virtually no DiI-LDL internalization with excess sterols and an ˜2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes. View Publication

Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore,this study aimed to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model. To evaluate their antigenicity and neutralization profile,indirect enzyme-linked immunosorbent assay (ELISA),an immunofluorescence assay (IFA) and a plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated,and reverse genetics techniques were then used to create an infectious CHIKV clone with a single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. The survival rate,CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralize CHIKV replication. Further analyses of 3E7b binding and neutralization of CHIKV single-mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In a pre-binding neutralization assay,3E7b blocks CHIKV attachment to permissive cells,possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection,3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively,these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design. View Publication