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Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb,helper-dependent adenoviral vectors with long homology arms are used for gene editing. However,this makes vector construction and recombinant analysis difficult. Conversely,insufficient homology may compromise targeting efficiency. Thus,we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation. With 23.8 and 21.4 kb of homology,the frequencies of targeted recombinants were 50-64.6% after positive selection for vector integration,and 97.4-100% after negative selection against random integrations. With 14.8 kb,the frequencies were 26.9-57.1% after positive selection and 87.5-100% after negative selection. With 9.6 kb,the frequencies were 21.4 and 75% after positive and negative selection,respectively. With only 5.6 kb,the frequencies were 5.6-16.7% after positive selection and 50% after negative selection,but these were more than high enough for efficient identification and isolation of targeted clones. Furthermore,we demonstrate helper-dependent adenoviral vector-mediated footprintless correction of cystic fibrosis transmembrane conductance regulator mutations through piggyBac excision of the selectable marker. However,low frequencies (≤ 1 × 10(-3)) necessitated negative selection for piggyBac-excision product isolation. View Publication

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines,of which a significantly higher ALDH activity was present in the OS99-1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99-1 cells were grown in NOD/SCID mice,we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDH(br) cells) from the OS99-1 xenografts were much less frequent,averaging 3% of the entire tumor population,compared to those isolated directly from the OS99-1 cell line. ALDH(br) cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDH(lo) cells),generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDH(br) cells illustrated the stem cell characteristics of self-renewal,the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A,Nanog and Sox-2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma-initiating cells and may potentially have therapeutic implications for human osteosarcoma. View Publication

Here we describe the in vitro generation of a novel adherent cell fraction derived from highly enriched,mobilized CD133(+) peripheral blood cells after their culture with Flt3/Flk2 ligand and interleukin-6 for 3 to 5 weeks. These cells lack markers of hematopoietic stem cells,endothelial cells,mesenchymal cells,dendritic cells,and stromal fibroblasts. However,all adherent cells expressed the adhesion molecules VE-cadherin,CD54,and CD44. They were also positive for CD164 and CD172a (signal regulatory protein-alpha) and for a stem cell antigen defined by the recently described antibody W7C5. Adherent cells can either spontaneously or upon stimulation with stem cell factor give rise to a transplantable,nonadherent CD133(+)CD34(-) stem cell subset. These cells do not generate in vitro hematopoietic colonies. However,their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice induced substantially higher long-term multilineage engraftment compared with that of freshly isolated CD34(+) cells,suggesting that these cells are highly enriched in SCID-repopulating cells. In addition to cells of the myeloid lineage,nonadherent CD34(-) cells were able to give rise to human cells with B-,T-,and natural killer-cell phenotype. Hence,these cells possess a distinct in vivo differentiation potential compared with that of CD34(+) stem cells and may therefore provide an alternative to CD34(+) progenitor cells for transplantation. View Publication

In an attempt to bring pluripotent stem cell biology closer to reaching its full potential,many groups have focused on improving reprogramming protocols over the past several years. The episomal modified Sendai virus-based vector has emerged as one of the most practical ones. Here we describe reprogramming of mesenchymal stromal/stem cells (MSC) derived from umbilical cord Wharton's Jelly into induced pluripotent stem cells (iPSC) using genome non-integrating Sendai virus-based vectors. The detailed protocols of iPSC colony cryopreservation (vitrification) and adaption to feeder-free culture conditions are also included. View Publication

Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs),but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs,sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (KATP) channels play crucial roles in excitability and cardioprotection. In this study,we aim to investigate the biological roles and use of sarcKATP and mitoKATP in hESC-VCM. We showed that SarcIK,ATP in single hESC-VCMs was dormant under baseline conditions,but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ˜8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly,sarcIK,ATP displayed a ˜3-fold increase after treatment with hypoxia (5% O2). MitoIK,ATP was absent in hESC-VCMs. However,the thyroid hormone T3 up-regulated mitoIK,ATP,conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system,T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O2). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoIK,ATP. View Publication

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II,the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature,fetal liver-like erythroblasts,that are smaller,express mostly fetal hemoglobin,and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated,and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary,because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA. View Publication

The purpose of this study was to examine the in vitro and in vivo osteogenic potential of human induced pluripotent stem cells (hiPSCs) against that of human bone marrow mesenchymal stem cells (hBMMSCs). Embryoid bodies (EBs),which were formed from undifferentiated hiPSCs,were dissociated into single cells and underwent osteogenic differentiation using the same medium as hBMMSCs for 14 days. Osteoinduced hiPSCs were implanted on the critical-size calvarial defects and long bone segmental defects in rats. The healing of defects was evaluated after 8 weeks and 12 weeks of implantation,respectively. Osteoinduced hiPSCs showed relatively lower and delayed in vitro expressions of the osteogenic marker COL1A1 and bone sialoprotein,as well as a weaker osteogenic differentiation through alkaline phosphatase staining and mineralization through Alizarin red staining compared with hBMMSCs. Calvarial defects treated with osteoinduced hiPSCs had comparable quality of new bone formation,including full restoration of bone width and robust formation of trabeculae,to those treated with hBMMSCs. Both osteoinduced hiPSCs and hBMMSCs persisted in regenerated bone after 8 weeks of implantation. In critical-size long bone segmental defects,osteoinduced hiPSC treatment also led to healing of segmental defects comparable to osteoinduced hBMMSC treatment after 12 weeks. In conclusion,despite delayed in vitro osteogenesis,hiPSCs have an in vivo osteogenic potential as good as hBMMSCs. View Publication

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs,composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive),endocrine precursors (NKX2.2/synaptophysin-positive,hormone/NKX6.1-negative),and polyhormonal cells (insulin/glucagon-positive,NKX6.1-negative). However,the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional β-cells remained unclear. To address this question,we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant,both populations contained a high proportion of PDX1-expressing cells (˜85%-90%) but were distinguished by their relatively high (˜80%) or low (˜25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study,but only NKX6.1-high grafts displayed robust meal-,glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue,but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells,whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high,but not NKX6.1-low grafts expressed nuclear MAFA. Collectively,this study demonstrates that a pancreatic endoderm-enriched population can mature into highly functional β-cells with only a minor contribution from the endocrine subpopulation. View Publication

BACKGROUND Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs),but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches,but the global proteome has not been examined. Furthermore,most hESC-CM studies focus on pathways important for cardiac differentiation,rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs,hESC-derived VCMs,human fetal and human adult ventricular and atrial CMs. METHODS AND RESULTS Using two-dimensional-differential-in-gel electrophoresis,121 differentially expressed (textgreater1.5-fold; Ptextless0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator-activated receptor $\$ in cardiac maturation. Consistently,WY-14643,a peroxisome proliferator-activated receptor $\$,increased fatty oxidative enzyme level,hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line,treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold,signifying their maturation. CONCLUSIONS We conclude that the peroxisome proliferator-activated receptor $\$ thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs. View Publication

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here,we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution,embryogenesis,and human disease,interspecies blastocyst complementation might allow human organ generation in animals whose organ size,anatomy,and physiology are closer to humans. To date,however,whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals,the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead,an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos. View Publication