Targeted biallelic integration of an inducible Caspase 9 suicide gene in iPSCs for safer therapies.

Drug-inducible suicide systems may help to minimize risks of human induced pluripotent stem cell (hiPSC) therapies. Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed. We have introduced a drug-inducible Caspase 9 suicide system (iCASP9) into the AAVS1 safe-harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. After transplantation into mice, drug treatment generally led to rapid elimination of teratomas, but single animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ∼ 3 × 10-8. Besides transgene elimination, presumably via loss of heterozygosity (LoH), silencing via aberrant promoter methylation was identified as a major underlying mechanism. In contrast to monoallelic iCASP9 hiPSCs, no escapees from biallelic iCASP9 cells were observed after treatment of up to 0.8 billion hiPSCs. The highly increased safety level provided by biallelic integration of the iCASP9 system may substantially contribute to the safety level of iPSC-based therapies.

Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes.

Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.

Targeted Integration of Inducible Caspase-9 in Human iPSCs Allows Efficient in vitro Clearance of iPSCs and iPSC-Macrophages.

Induced pluripotent stem cells (iPSCs) offer great promise for the field of regenerative medicine, and iPSC-derived cells have already been applied in clinical practice. However, potential contamination of effector cells with residual pluripotent cells (e.g., teratoma-initiating cells) or effector cell-associated side effects may limit this approach. This also holds true for iPSC-derived hematopoietic cells. Given the therapeutic benefit of macrophages in different disease entities and the feasibility to derive macrophages from human iPSCs, we established human iPSCs harboring the inducible Caspase-9 (iCasp9) suicide safety switch utilizing transcription activator-like effector nuclease (TALEN)-based designer nuclease technology. Mono- or bi-allelic integration of the iCasp9 gene cassette into the AAVS1 locus showed no effect on the pluripotency of human iPSCs and did not interfere with their differentiation towards macrophages. In both, iCasp9-mono and iCasp9-bi-allelic clones, concentrations of 0.1 nM AP20187 were sufficient to induce apoptosis in more than 98% of iPSCs and their progeny-macrophages. Thus, here we provide evidence that the introduction of the iCasp9 suicide gene into the AAVS1 locus enables the effective clearance of human iPSCs and thereof derived macrophages.

Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture.

Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding ∼1.3 × 108 CM in 150 mL and ∼4.0 × 108 CMs in 350-500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives.

Induction of pluripotent stem cells from a cynomolgus monkey using a polycistronic simian immunodeficiency virus-based vector, differentiation toward functional cardiomyocytes, and generation of stably expressing reporter lines.

Induced pluripotent stem cells (iPSCs) represent a novel cell source for regenerative therapies. Many emerging iPSC-based therapeutic concepts will require preclinical evaluation in suitable large animal models. Among the large animal species frequently used in preclinical efficacy and safety studies, macaques show the highest similarities to humans at physiological, cellular, and molecular levels. We have generated iPSCs from cynomolgus monkeys (Macaca fascicularis) as a segue to regenerative therapy model development in this species. Because typical human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors show poor transduction of simian cells, a simian immunodeficiency virus (SIV)-based vector was chosen for efficient transduction of cynomolgus skin fibroblasts. A corresponding polycistronic vector with codon-optimized reprogramming factors was constructed for reprogramming. Growth characteristics as well as cell and colony morphology of the resulting cynomolgus iPSCs (cyiPSCs) were demonstrated to be almost identical to cynomolgus embryonic stem cells (cyESCs), and cyiPSCs expressed typical pluripotency markers including OCT4, SOX2, and NANOG. Furthermore, differentiation in vivo and in vitro into derivatives of all three germ layers, as well as generation of functional cardiomyocytes, could be demonstrated. Finally, a highly efficient technique for generation of transgenic cyiPSC clones with stable reporter expression in undifferentiated cells as well as differentiated transgenic cyiPSC progeny was developed to enable cell tracking in recipient animals. In conclusion, our data indicate that cyiPSCs represent a valuable cell source for establishment of macaque-based allogeneic and autologous preclinical cell transplantation models for various fields of regenerative medicine.

Generation of induced pluripotent stem cells from human cord blood.

Induced pluripotent stem cells (iPSCs) may represent an ideal cell source for future regenerative therapies. A critical issue concerning the clinical use of patient-specific iPSCs is the accumulation of mutations in somatic (stem) cells over an organism's lifetime. Acquired somatic mutations are passed onto iPSCs during reprogramming and may be associated with loss of cellular functions and cancer formation. Here we report the generation of human iPSCs from cord blood (CB) as a juvenescent cell source. CBiPSCs show characteristics typical of embryonic stem cells and can be differentiated into derivatives of all three germ layers, including functional cardiomyocytes. For future therapeutic production of autologous and allogeneic iPSC derivatives, CB could be routinely harvested for public and commercial CB banks without any donor risk. CB could readily become available for pediatric patients and, in particular, for newborns with genetic diseases or congenital malformations.

Human CMV immediate-early enhancer: a useful tool to enhance cell-type-specific expression from lentiviral vectors.

BACKGROUND: Lentiviral vectors are attractive delivery tools for gene therapy, especially in terminally differentiated target cells. While restriction of gene expression to specific cell populations is of particular importance, highly efficient cell-type-specific gene expression after viral gene transfer so far has been hampered by low levels of transgene expression. METHODS: Addressing this problem, we have integrated the human cytomegalovirus (CMV) immediate-early enhancer into an 'advanced' generation lentiviral vector. Expression cassettes with the reporter gene green fluorescent protein (GFP), combined with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) under control of a ubiquitous phosphoglycerate kinase (mouse PGK), cardiomyocyte- (human atrial natriuretic factor (ANF), human ventricular myosin light chain (MLC2v)), or type II alveolar epithelial cell (AT-2)-specific human surfactant protein C (SP-C) promoter, were introduced. As insertion of an enhancing element can interfere with the promoter's specificity, expression levels conferred by our enhancer/promoter constructs were evaluated in target and non-target cells. RESULTS: Transduction of target cells with human CMV enhancer containing lentiviral vectors resulted in a multiple-log increase in GFP expression compared to corresponding vectors lacking the human CMV enhancer. In the case of the ANF, the MLC2v, and the SP-C promoters, tissue-specific reporter gene expression in cardiomyocytes and in lung AT-2 cells was maintained, as expression in non-target cells increased only up to 7-fold. CONCLUSIONS: The results of this study indicate that lentiviral vectors with the human CMV enhancer conferring efficient cell-type-specific gene expression may be useful tools for gene therapy purposes or cell tracing, e.g. to analyze stem cell differentiation in transplantation and co-culture settings.

Type II pneumocyte-restricted green fluorescent protein expression after lentiviral transduction of lung epithelial cells.

Type II alveolar epithelial (AT2) cell-specific reporter expression has been highly useful in the study of embryology and alveolar regeneration in transgenic mice. Technologies enabling efficient gene transfer and cell type-restricted transgene expression in AT2 cells would allow for correction of AT2 cell-based diseases such as genetic surfactant deficiencies. Moreover, such approaches are urgently required to investigate differentiation of AT2 cells from adult and embryonic stem cells of other species than mouse. Using a human surfactant protein C (SP-C) promoter fragment, we have constructed lentiviral vectors enabling AT2-restricted transgene expression and identification of stem cell-derived AT2 cells. Lung epithelial cell lines M3E3/C3, H441, RLE-6TN, A549, MLE-12, and MLE-15 were characterized at the molecular and ultrastructural levels to identify cell lines useful to assess the cell type specificity of our vector constructs. After transduction, no green fluorescent protein (GFP) expression was observed in nontarget cells including bronchial H441 cells, pulmonary A549 cells, fibroblasts, smooth muscle cells, and endothelial cells. In contrast, and in correlation with endogenous SP-C expression, lentiviral transduction resulted in stable GFP expression in MLE-12 and MLE-15 AT2 cells. In conclusion, we have constructed a lentiviral vector mediating SP-C promoter-dependent GFP expression. Transgene expression strictly corresponds with an AT2 phenotype of the transduced cells. In particular, the generated vector should facilitate local alveolar gene therapy and investigation of alveolar regeneration and stem cell differentiation.

Generation and characterization of functional cardiomyocytes from rhesus monkey embryonic stem cells.

Embryonic stem cells (ESCs) from mice and humans (hESCs) have been shown to be able to efficiently differentiate toward cardiomyocytes (CMs). Because murine ESCs and hESCs do not allow for establishment of pre-clinical allogeneic transplantation models, the aim of our study was to generate functional CMs from rhesus monkey ESCs (rESCs). Although formation of ectodermal and neuronal/glial cells appears to be the default pathway of the rESC line R366.4, we were able to change this commitment and to direct generation of endodermal/mesodermal cells and further differentiation toward CMs. Differentiation of rESCs resulted in an average of 18% of spontaneously contracting embryoid bodies (EBs) from rESCs. Semiquantitative reverse transcription-polymerase chain reaction analyses demonstrated expression of marker genes typical for endoderm, mesoderm, cardiac mesoderm, and CMs, including brachyury, goosecoid, Tbx-5, Tbx-20, Mesp1, Nkx2.5, GATA-4, FOG-2, Mlc2a, MLC2v, ANF, and alpha-MHC in rESC-derived CMs. Immunohistological and ultrastructural studies showed expression of CM-typical proteins, including sarcomeric actinin, troponin T, titin, connexin 43, and cross-striated muscle fibrils. Electrophysiological studies by means of multielectrode arrays revealed evidence of functionality, electrical coupling, and beta-adrenergic signaling of the generated CMs. This is the first study demonstrating generation of functional CMs derived from rESCs. In contrast to hESCs, rESCs allow for establishment of pre-clinical allogeneic transplantation models. Moreover, rESC-derived CMs represent a cell source for the development of high-throughput assays for cardiac safety pharmacology.