Zhaohui Ye

Gene Targeting of a Disease-Related Gene in Human Induced Pluripotent Stem and Embryonic Stem Cells

We report here homologous recombination (HR)-mediated gene targeting of two different genes in human iPS cells (hiPSCs) and human ES cells (hESCs). HR-mediated correction of a chromosomally integrated mutant GFP reporter gene reaches efficiencies of 0.14%-0.24% in both cell types transfected by donor DNA with plasmids expressing zinc finger nucleases (ZFNs). Engineered ZFNs that induce a sequence-specific double-strand break in the GFP gene enhanced HR-mediated correction by > 1400-fold without detectable alterations in stem cell karyotypes or pluripotency. Efficient HR-mediated insertional mutagenesis was also achieved at the endogenous PIG-A locus, with a > 200-fold enhancement by ZFNs targeted to that gene. Clonal PIG-A null hESCs and iPSCs with normal karyotypes were readily obtained. The phenotypic and biological defects were rescued by PIG-A transgene expression. Our study provides the first demonstration of HR-mediated gene targeting in hiPSCs and shows the power of ZFNs for inducing specific genetic modifications in hiPSCs, as well as hESCs.

Human Adult Marrow Cells Support Prolonged Expansion of Human Embryonic Stem Cells in Culture

Prolonged propagation of human embryonic stem (hES) cells is currently achieved by coculture with primary mouse embryonic fibroblasts (MEFs) serving as feeder cells. Unlike mouse ES cells, adding growth factors such as leukemia inhibitory factor is insufficient to maintain undifferentiated hES cells without feeder cells. The presence of uncharacterized rodent cells or crude extracts imposes a risk to the clinical applications of hES cells. While others looked for a replacement of MEFs with human fetal cells, we attempted to use easily accessible postnatal human cells such as human marrow stromal cells (hMSCs). Culture‐expanded hMSCs from multiple donors were used as feeder cells to support growth of the H1 hES cell line under a serum‐free culture condition. Human ES cell colonies cultured on irradiated hMSCs amplified >100‐fold during the 30‐day continuous culture (in five passages). The longest continuous expansion of hES cells on hMSCs tested to date is 13 passages. The expanded hES cells displayed the unique morphology and molecular markers characteristic of undifferentiated hES cells as observed when they were cultured on MEFs. They expressed the transcription factor Oct‐4, a membrane alkaline phosphatase, and the stage‐specific embryonic antigen (SSEA)‐4, but not the SSEA‐1 marker. Expanded hES cells on hMSCs retained unique differentiation potentials in culture and a normal diploid karyotype. The well‐studied hMSCs (and this animal cell‐ and serum‐free system) may provide a clinically and ethically feasible method to expand hES cells for novel cell therapies. In addition, this system may help to identify cytokines and adhesion molecules that are required for the self‐renewal of hES cells.

Butyrate Greatly Enhances Derivation of Human Induced Pluripotent Stem Cells by Promoting Epigenetic Remodeling and the Expression of Pluripotency-Associated Genes

We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15‐ to 51‐fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100‐ to 200‐fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome‐wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency‐associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming. STEM CELLS 2010;28:713–72028:713–720

Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34(+) cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34(+) cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34(+)CD45(+)) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34(+) cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

Defining the Role of Wnt/β-Catenin Signaling in the Survival, Proliferation, and Self-Renewal of Human Embryonic Stem Cells

We used a panel of human and mouse fibroblasts with various abilities for supporting the prolonged growth of human embryonic stem cells (hESCs) to elucidate growth factors required for hESC survival, proliferation, and maintenance of the undifferentiated and pluripotent state (self‐renewal). We found that supportive feeder cells secrete growth factors required for both hESC survival/proliferation and blocking hESC spontaneous differentiation to achieve self‐renewal. The antidifferentiation soluble factor is neither leukemia inhibitory factor nor Wnt, based on blocking experiments using their antagonists. Because Wnt/β‐catenin signaling has been implicated in cell‐fate determination and stem cell expansion, we further examined the effects of blocking or adding recombinant Wnt proteins on undifferentiated hESCs. In the absence of feeder cell–derived factors, hESCs cultured under a feeder‐free condition survived/proliferated poorly and gradually differentiated. Adding recombinant Wnt3a stimulated hESC proliferation but also differentiation. After 4–5 days of Wnt3a treatment, hESCs that survived maintained the undifferentiated phenotype but few could form undifferentiated hESC colonies subsequently. Using a functional reporter assay, we found that the β‐catenin–mediated transcriptional activation in the canonical Wnt pathway was minimal in undifferentiated hESCs, but greatly upregulated during differentiation induced by the Wnt treatment and several other methods. Thus, Wnt/β‐catenin activation does not suffice to maintain the undifferentiated and pluripotent state of hESCs. We propose a new model for the role of Wnt/β‐catenin signaling in undifferentiated hESCs.

Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures

To identify accessible and permissive human cell types for efficient derivation of induced pluripotent stem cells (iPSCs), we investigated epigenetic and gene expression signatures of multiple postnatal cell types such as fibroblasts and blood cells. Our analysis suggested that newborn cord blood (CB) and adult peripheral blood (PB) mononuclear cells (MNCs) display unique signatures that are closer to iPSCs and human embryonic stem cells (ESCs) than age-matched fibroblasts to iPSCs/ESCs, thus making blood MNCs an attractive cell choice for the generation of integration-free iPSCs. Using an improved EBNA1/OriP plasmid expressing 5 reprogramming factors, we demonstrated highly efficient reprogramming of briefly cultured blood MNCs. Within 14 days of one-time transfection by one plasmid, up to 1000 iPSC-like colonies per 2 million transfected CB MNCs were generated. The efficiency of deriving iPSCs from adult PB MNCs was approximately 50-fold lower, but could be enhanced by inclusion of a second EBNA1/OriP plasmid for transient expression of additional genes such as SV40 T antigen. The duration of obtaining bona fide iPSC colonies from adult PB MNCs was reduced to half (∼14 days) as compared to adult fibroblastic cells (28–30 days). More than 9 human iPSC lines derived from PB or CB blood cells are extensively characterized, including those from PB MNCs of an adult patient with sickle cell disease. They lack V(D)J DNA rearrangements and vector DNA after expansion for 10–12 passages. This facile method of generating integration-free human iPSCs from blood MNCs will accelerate their use in both research and future clinical applications.

Improved Efficiency and Pace of Generating Induced Pluripotent Stem Cells from Human Adult and Fetal Fibroblasts

It was reported recently that human fibroblasts can be reprogrammed into a pluripotent state that resembles that of human embryonic stem (hES) cells. This was achieved by ectopic expression of four genes followed by culture on mouse embryonic fibroblast (MEF) feeders under a condition favoring hES cell growth. However, the efficiency of generating human induced pluripotent stem (iPS) cells is low, especially for postnatal human fibroblasts. We started supplementing with an additional gene or bioactive molecules to increase the efficiency of generating iPS cells from human adult as well as fetal fibroblasts. We report here that adding SV40 large T antigen (T) to either set of the four reprogramming genes previously used enhanced the efficiency by 23–70‐fold from both human adult and fetal fibroblasts. Discernible hES‐like colonies also emerged 1–2 weeks earlier if T was added. With the improved efficiency, we succeeded in replacing MEFs with immortalized human feeder cells that we previously established for optimal hES cell growth. We further characterized individually picked hES‐like colonies after expansion (up to 24 passages). The majority of them expressed various undifferentiated hES markers. Some but not all the hES‐like clones can be induced to differentiate into the derivatives of the three embryonic germ layers in both teratoma formation and embryoid body (EB) formation assays. These pluripotent clones also differentiated into trophoblasts after EB formation or bone morphogenetic protein 4 induction as classic hES cells. Using this improved approach, we also generated hES‐like cells from homozygous fibroblasts containing the sickle cell anemia mutation Hemoglobin Sickle.

In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells

Development of clinically relevant regenerative medicine therapies using human embryonic stem cells (hESCs) requires production of a simple and readily expandable cell population that can be directed to form functional 3D tissue in an in vivo environment. We describe an efficient derivation method and characterization of mesenchymal stem cells (MSCs) from hESCs (hESCd-MSCs) that have multilineage differentiation potential and are capable of producing fat, cartilage, and bone in vitro. Furthermore, we highlight their in vivo survival and commitment to the chondrogenic lineage in a microenvironment comprising chondrocyte-secreted morphogenetic factors and hydrogels. Normal cartilage architecture was established in rat osteochondral defects after treatment with chondrogenically-committed hESCd-MSCs. In view of the limited available cell sources for tissue engineering applications, these embryonic-derived cells show significant potential in musculoskeletal tissue regeneration applications.

Whole-Genome Sequencing Analysis Reveals High Specificity of CRISPR/Cas9 and TALEN-Based Genome Editing in Human iPSCs

Human iPSCs provide renewable cell sources for human biology and disease research and the potential for developing gene and cell therapy. Realization of this potential will rely in part on our ability to precisely edit or engineer the human genome in an efficient way. Recent developments in designer endonuclease technologies such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease have provided ways to significantly improve genome editing efficiency in human iPSCs. These endonucleases make a double-stranded break (DSB) at a predetermined DNA sequence and trigger natural DNA repair processes such as nonhomologous end joining (NHEJ) or homologous recombination (HR) with a donor DNA template. Among these existing approaches, RNA-guided CRISPR/Cas9 is the most user-friendly and versatile system, and it has been applied in both animal models and cell lines (Cong et al., 2013; Hsu et al., 2014; Mali et al., 2013). The most commonly used system consists of a single polypeptide endonuclease Cas9 complexed with a single guide RNA (gRNA) that provides complementarity to 20-nucleotide target DNA sequence. However, the specificity and efficiency of this approach in human iPSCs have not been studied in detail (Cong et al., 2013; Ding et al., 2013; Mali et al., 2013; Yang et al., 2013). Some analyses using cancer cell lines reported higher-than-expected levels of off-target mutagenesis by Cas9-gRNAs (Fu et al., 2013; Hsu et al., 2013), raising concerns about the practical applicability of this approach in therapeutic contexts. Some recent studies, including one on human adult stem cells, showed a minimal level of off-target effects by CRISPR/Cas9 (Schwank et al., 2013). However, these existing analyses of off-target effects and mutational load in gene-corrected stem cells have been restricted to checking predicted off target sites and are therefore limited in scope. To assess the value of this type of gene editing approach for therapeutic applications, it is critical to rigorously examine whether it is possible to generate gene-edited cell lines with minimal mutational load. To this end, we have conducted whole-genome sequencing of four iPSC clones successfully targeted at the AAVS1 locus, a “safe harbor” in the human genome that is used for stable transgene expression in a variety of contexts. To generate the lines, we used an integration-free human iPSC line, BC1, whose genomic integrity has been characterized in detail by next-generation sequencing (Cheng et al., 2012) and targeted a GFP expression cassette into the AAVS1 site with either a previously reported Cas9-gRNA combination or a pair of improved heterodimeric TALENs (Mali et al., 2013; Yan et al., 2013) (Table S1 and Supplemental Experimental Procedures available online). Twenty days after transfection of the donor plasmid and either the TALENs or Cas9-gRNA into BC1, we harvested four clones with confirmed targeted integration (hCas9-C4, hCas9-C16, TALEN-C3, and TALEN-C6; Table S1 and Supplemental Experimental Procedures) and the parental BC1 iPSCs for whole-genome sequencing. The sequencing reads, ranging from 83 Gbps to 100 Gbps from each targeted clone, were first aligned to the human hg19 reference genome to enable identification of single-nucleotide variants (SNVs) and small indels (Table S1). Our analysis identified ≥4.2 million SNVs and ≥500,000 indels in each genome (Table S1) in comparison to the hg19 reference genome, suggesting that it is a rigorous data set that covers the genome in sufficient depth to detect sequence variants. The “germ-line” variants (present in BC1 parental iPSCs and different from hg19) were readily detectable in each targeted cell line (80%%–88%), indicating that the sensitivity of variant detection in our analysis is high (Table S1). The variations from each targeted clone were then compared to the BC1 parental iPSCs to enable the generation of a list of potential variations arising during the gene editing process, which we then confirmed using genomic PCR and Sanger sequencing. We confirmed 62 out of 69 SNVs tested for an overall confirmation rate of 90%, and based on that we estimate that the total SNVs in the four iPSC clones range between 217 and 281 and that the total indels range between 7 and 12 (Table S1). Overall the genomic variation levels in TALEN- and Cas9-targeted groups were comparable. One important consideration is how many of these detected SNVs and indels were the results of off-target mutagenesis by the engineered endonucleases. To address this question, we generated a list of 3,665 (Cas9) and 238 (TALEN) putative off-target positions by using the EMBOSS fuzznuc software package. Each candidate SNV and indel was compared to this list and none of them are within a potential off-target region (Table S1), consistent with previous analyses looking at predicted off-target sites. Our analysis also shows that each SNV and indel is unique and that none of them occurred in more than one cell line. The absence of recurring mutations and the fact that none of the mutations resides in any putative off-target site by bioinformatic prediction strongly suggest that these mutations were randomly accumulated during regular cell expansion and are not direct results of off-target activities by Cas9 or TALENs. Our results from whole-genome sequencing analysis of Cas9- and TALEN-targeted human iPSC clones demonstrate that these engineered endonucleases provide efficient genome-editing tools with high specificity. It remains to be clarified whether the higher off-target rates observed in cancer cell lines are due to the overexpression of gRNAs and Cas9 protein and/or due to exacerbated and faulty DNA repair in these cell types. The higher specificity observed in human iPSCs, combined with the rapid development of next-generation sequencing technology, makes it possible to characterize and isolate high quality genome-edited stem cell clones with minimal mutational load. The guiding principle established with human iPSCs will likely be applicable to other types of stem cells and come with improvements in gene transfer and targeting efficiencies. Our current study of gene targeting in human iPSCs will help to establish better models for human biology and disease research and to provide proof-of-principle for future gene therapy.

Generation of endoderm‐derived human induced pluripotent stem cells from primary hepatocytes

Recent advances in induced pluripotent stem (iPS) cell research have significantly changed our perspective on regenerative medicine. Patient‐specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy. However, there have been insufficient data to prove that iPS cells are functionally equivalent to human embryonic stem (hES) cells or are safer than hES cells. There are several important issues that need to be addressed, and foremost are the safety and efficacy of human iPS cells of different origins. Human iPS cells have been derived mostly from cells originating from mesoderm and in a few cases from ectoderm. So far, there has been no report of endoderm–derived human iPS cells, and this has prevented comprehensive comparative investigations of the quality of human iPS cells of different origins. Here we show for the first time reprogramming of human endoderm‐derived cells (i.e., primary hepatocytes) to pluripotency. Hepatocyte‐derived iPS cells appear indistinguishable from hES cells with respect to colony morphology, growth properties, expression of pluripotency‐associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells are able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. Conclusion: The technology to develop endoderm–derived human iPS cell lines, together with other established cell lines, will provide a foundation for elucidating the mechanisms of cellular reprogramming and for studying the safety and efficacy of differentially originated human iPS cells for cell therapy. For the study of liver disease pathogenesis, this technology also provides a potentially more amenable system for generating liver disease‐specific iPS cells. (HEPATOLOGY 2010;51:1810–1819)