A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach.

Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications.

Differential sensitivity to JAK inhibitory drugs by isogenic human erythroblasts and hematopoietic progenitors generated from patient-specific induced pluripotent stem cells.

Disease-specific induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to establish novel disease models and accelerate drug development using distinct tissue target cells generated from isogenic iPSC lines with and without disease-causing mutations. To realize the potential of iPSCs in modeling acquired diseases which are usually heterogeneous, we have generated multiple iPSC lines including two lines that are JAK2-wild-type and four lines homozygous for JAK2-V617F somatic mutation from a single polycythemia vera (PV) patient blood. In vitro differentiation of the same patient-derived iPSC lines have demonstrated the differential contributions of their parental hematopoietic clones to the abnormal erythropoiesis including the formation of endogenous erythroid colonies. This iPSC approach thus may provide unique and valuable insights into the genetic events responsible for disease development. To examine the potential of iPSCs in drug testing, we generated isogenic hematopoietic progenitors and erythroblasts from the same iPSC lines derived from PV patients and normal donors. Their response to three clinical JAK inhibitors, INCB018424 (Ruxolitinib), TG101348 (SAR302503), and the more recent CYT387 was evaluated. All three drugs similarly inhibited erythropoiesis from normal and PV iPSC lines containing the wild-type JAK2 genotype, as well as those containing a homozygous or heterozygous JAK2-V617F activating mutation that showed increased erythropoiesis without a JAK inhibitor. However, the JAK inhibitors had less inhibitory effect on the self-renewal of CD34+ hematopoietic progenitors. The iPSC-mediated disease modeling thus underlies the ineffectiveness of the current JAK inhibitors and provides a modeling system to develop better targeted therapies for the JAK2 mutated hematopoiesis.

Extensive ex vivo expansion of functional human erythroid precursors established from umbilical cord blood cells by defined factors.

There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10(6)-10(7)-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~10(68)-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine.

Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.

Several human postnatal somatic cell types have been successfully reprogrammed to induced pluripotent stem cells (iPSCs). Blood mononuclear cells (MNCs) offer several advantages compared with other cell types. They are easily isolated from umbilical cord blood (CB) or adult peripheral blood (PB), and can be used fresh or after freezing. A short culture allows for more efficient reprogramming, with iPSC colonies forming from blood MNCs in 14 d, compared with 28 d for age-matched fibroblastic cells. The advantages of briefly cultured blood MNCs may be due to favorable epigenetic profiles and gene expression patterns. Blood cells from adults, especially nonlymphoid cells that are replenished frequently from intermittently activated blood stem cells, are short-lived in vivo and may contain less somatic mutations than skin fibroblasts, which are more exposed to environmental mutagens over time. We describe here a detailed, validated protocol for effective generation of integration-free human iPSCs from blood MNCs by plasmid vectors.

Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by nonintegrating plasmid expression.

The utility of induced pluripotent stem cells (iPSCs) as models to study diseases and as sources for cell therapy depends on the integrity of their genomes. Despite recent publications of DNA sequence variations in the iPSCs, the true scope of such changes for the entire genome is not clear. Here we report the whole-genome sequencing of three human iPSC lines derived from two cell types of an adult donor by episomal vectors. The vector sequence was undetectable in the deeply sequenced iPSC lines. We identified 1,058-1,808 heterozygous single-nucleotide variants (SNVs), but no copy-number variants, in each iPSC line. Six to twelve of these SNVs were within coding regions in each iPSC line, but ~50% of them are synonymous changes and the remaining are not selectively enriched for known genes associated with cancers. Our data thus suggest that episome-mediated reprogramming is not inherently mutagenic during integration-free iPSC induction.

Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines.

Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease.

Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated ß-globin alleles (ß(s)/ß(s)). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the ß(s) locus, we achieved precise conversion of 1 mutated ß(s) to the wild-type ß(A) in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained "secondary" gene-corrected ß(s)/ß(A) heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.

Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting.

We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD), a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91(phox) deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production, reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs, with subsequent selection and full characterization to ensure no off-target changes, holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the "safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs.

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.