Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks.

A promising cell-therapy approach for heart failure aims at differentiating human pluripotent stem cells (hPSCs) into functional cardiomyocytes (CMs) in vitro to replace the disease-induced loss of patients' heart muscle cells in vivo. But many challenges remain for the routine clinical application of hPSC-derived CMs (hPSC-CMs), including good manufacturing practice (GMP)-compliant production strategies. This protocol describes the efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs. Using a simple spinner-flask platform, this protocol is applicable to a broad range of users with general experience in handling hPSCs without extensive know-how in biotechnology. hPSCs are expanded in monolayer to generate the required cell numbers for process inoculation in suspension culture, followed by stirring-controlled formation of cell-only aggregates at a 300-ml scale. After 48 h at checkpoint (CP) 0, chemically defined cardiac differentiation is induced by WNT-pathway modulation through use of the glycogen-synthase kinase-3 inhibitor CHIR99021 (WNT agonist), which is replaced 24 h later by the chemical WNT-pathway inhibitor IWP-2. The exact application of the described process parameters is important to ensure process efficiency and robustness. After 10 d of differentiation (CP I), the production of ≥100 × 106 CMs is expected. Moreover, to 'uncouple' cell production from downstream applications, continuous maintenance of CM aggregates for up to 35 d in culture (CP II) is demonstrated without a reduction in CM content, supporting downstream logistics while potentially overcoming the requirement for cryopreservation.

Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation.

BACKGROUND: Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown. METHODS AND RESULTS: STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs' pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls. CONCLUSIONS: Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.

Regulation of STING activity in DNA sensing by ISG15 modification.

Sensing of human immunodeficiency virus type 1 (HIV-1) DNA is mediated by the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling axis. Signal transduction and regulation of this cascade is achieved by post-translational modifications. Here we show that cGAS-STING-dependent HIV-1 sensing requires interferon-stimulated gene 15 (ISG15). ISG15 deficiency inhibits STING-dependent sensing of HIV-1 and STING agonist-induced antiviral response. Upon external stimuli, STING undergoes ISGylation at residues K224, K236, K289, K347, K338, and K370. Inhibition of STING ISGylation at K289 suppresses STING-mediated type Ⅰ interferon induction by inhibiting its oligomerization. Of note, removal of STING ISGylation alleviates gain-of-function phenotype in STING-associated vasculopathy with onset in infancy (SAVI). Molecular modeling suggests that ISGylation of K289 is an important regulator of oligomerization. Taken together, our data demonstrate that ISGylation at K289 is crucial for STING activation and represents an important regulatory step in DNA sensing of viruses and autoimmune responses.

Generation of an induced pluripotent stem cell line, ZIPi021-A, from fibroblasts of a Prader-Willi syndrome patient with maternal uniparental disomy (mUPD).

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by loss of paternal expression of imprinted genes on chromosome 15q11-q13. We established a human induced pluripotent stem cell line (hiPSC), ZIPi021-A, from fibroblasts of a 4-year-old female PWS patient with the subtype of maternal uniparental disomy (mUPD). The generated hiPSC line was transgene-free, expressed pluripotency markers and showed the ability to differentiate into all three germ layers in vitro. The ZIPi021-A hiPSC line could be used as a cellular model for PWS in humans.

Generation of two human NRF2 knockout iPSC clones using CRISPR/Cas9 editing.

The nuclear factor erythroid 2-related factor 2 (NFE2L2, known as NRF2) regulates the expression of antioxidative and anti-inflammatory proteins. In order to investigate its impact during viral infections and testing of antiviral compounds, we applied CRISPR/Cas9 editing to eliminate NRF2 in the human iPS cell line MHHi001-A and generated two NRF2 knockout iPSC clones MHHi001-A-6 and MHHi001-A-7. After differentiation into epithelia or endothelial cells, these cells are useful tools to examine the antiviral effects of activators of the NRF2 signaling pathway.

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.

Generation of human induced pluripotent stem cell lines encoding for genetically encoded calcium indicators RCaMP1h and GCaMP6f.

Calcium plays a key role in cardiomyocytes (CMs) for the translation of the electrical impulse of an action potential into contraction forces. A rapid, not-invasive fluorescence imaging technology allows for the monitoring of calcium transients in human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) to investigate the cardiac electrophysiology in vitro and after cell transplantation in vivo. The genetically encoded calcium indicators (GECIs) GCaMP6f or RCaMP1h were successfully transfected in the previously established hiPSC line MHHi001-A, together with a cardiac specific antibiotic selection cassette facilitating the monitoring of the calcium handling in highly pure populations of hiPSC-CMs.

A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines.

CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.

Generation of pulmonary arterial hypertension patient-specific induced pluripotent stem cell lines from three unrelated patients with a heterozygous missense mutation in exon 12, a heterozygous in-frame deletion in exon 3 and a missense mutation in exon 11 of the BMPR2 gene.

Loss-of-function mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene are common in heritable or idiopathic pulmonary arterial hypertension (PAH), and can result in functional impairment of both endothelial and vascular smooth muscle cells. Here, we report 3 PAH patient-specific induced pluripotent stem cells (iPSC) lines from 3 unrelated patients harbouring different mutations in the BMPR2 gene: a heterozygous missense mutation in exon 12, a heterozygous frame shift deletion in exon 3, and a heterozygous missense mutation in exon 11. These cell lines will serve as a valuable resource to model PAH in vitro.

Assessing the Single and Combined Toxicity of the Bioinsecticide Spear and Cry3Bb1 Protein Against Susceptible and Resistant Western Corn Rootworm Larvae (Coleoptera: Chrysomelidae).

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), poses a serious threat to maize (Zea mays L.) growers in the U.S. Corn Belt. Transgenic corn expressing Bacillus thuringiensis (Bt) Berliner is the major management tactic along with crop rotation. Bt crops targeting WCR populations have been widely planted throughout the Corn Belt. Rootworms have developed resistance to nearly all management strategies including Bt corn. Therefore, there is a need for new products that are not cross-resistant with the current Bt proteins. In this study, we evaluated the susceptibility of WCR strains resistant and susceptible to Cry3Bb1 to the biological insecticide Spear-T (GS-omega/kappa-Hexatoxin-Hv1a) alone and combined with Cry3Bb1 protein. The activity of Hv1a alone was similar between Cry3Bb1-resistant and susceptible strains (LC50s = 0.95 mg/cm2 and 1.50 mg/cm2, respectively), suggesting that there is no cross-resistance with Cry3Bb1 protein. Effective concentration (EC50), molt inhibition concentration (MIC50), and inhibition concentration (IC50) values of Hv1a alone were also similar between both strains, based on non-overlapping confidence intervals. Increased mortality (64%) was observed on resistant larvae exposed to Hv1a (0.6 mg/cm2) + Cry3Bb1 protein (170.8 µg/cm2) compared to 0% mortality when exposed to Cry3Bb1 alone and 34% mortality to Hv1a alone (0.3 mg/cm2). The time of larval death was not significantly different between Hv1a alone (3.79 mg/cm2) and Hv1a (0.6 mg/cm2) + Cry3Bb1 (170.8 µg/cm2). New control strategies that are not cross-resistant with current insecticides and Bt proteins are needed to better manage the WCR, and Hv1a together with Cry3Bb1 may fit this role.

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.

High density bioprocessing of human pluripotent stem cells by metabolic control and in silico modeling.

To harness the full potential of human pluripotent stem cells (hPSCs) we combined instrumented stirred tank bioreactor (STBR) technology with the power of in silico process modeling to overcome substantial, hPSC-specific hurdles toward their mass production. Perfused suspension culture (3D) of matrix-free hPSC aggregates in STBRs was applied to identify and control process-limiting parameters including pH, dissolved oxygen, glucose and lactate levels, and the obviation of osmolality peaks provoked by high density culture. Media supplements promoted single cell-based process inoculation and hydrodynamic aggregate size control. Wet lab-derived process characteristics enabled predictive in silico modeling as a new rational for hPSC cultivation. Consequently, hPSC line-independent maintenance of exponential cell proliferation was achieved. The strategy yielded 70-fold cell expansion in 7 days achieving an unmatched density of 35 × 106 cells/mL equivalent to 5.25 billion hPSC in 150 mL scale while pluripotency, differentiation potential, and karyotype stability was maintained. In parallel, media requirements were reduced by 75% demonstrating the outstanding increase in efficiency. Minimal input to our in silico model accurately predicts all main process parameters; combined with calculation-controlled hPSC aggregation kinetics, linear process upscaling is also enabled and demonstrated for up to 500 mL scale in an independent bioreactor system. Thus, by merging applied stem cell research with recent knowhow from industrial cell fermentation, a new level of hPSC bioprocessing is revealed fueling their automated production for industrial and therapeutic applications.

Establishment of MHHi001-A-5, a GCaMP6f and RedStar dual reporter human iPSC line for in vitro and in vivo characterization and in situ tracing of iPSC derivatives.

Transgenic hiPSC lines carrying reporter genes represent valuable tools for functional characterization of iPSC derivatives, disease modelling and clinical evaluation of cell therapies. Here, the hiPSC line 'Phoenix' (Haase et al., 2017) was genetically engineered using TALEN-based integration of the calcium sensor GCaMP6f and RedStarnuc reporter into the AAVS1 site. Characterization of undifferentiated cells and functional investigation of hiPSC-derived cardiomyocytes-containing BCTs showed a strong intracellular calcium transient-dependent GCaMP6f and eminent RedStarnuc signal. Therefore, our dual reporter line provides an excellent tool to facilitate monitoring of engraftment, calcium fluctuations and coupling of iPSC derivatives such as cardiomyocytes in vitro and in vivo in animal models.

Increased prostaglandin-D2 in male STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation.

Cardiac levels of the signal transducer and activator of transcription factor-3 (STAT3) decline with age, and male but not female mice with a cardiomyocyte-specific STAT3 deficiency conditional knockout (CKO) display premature age-related heart failure associated with reduced cardiac capillary density. In the present study, isolated male and female CKO-cardiomyocytes exhibit increased prostaglandin (PG)-generating cyclooxygenase-2 (COX-2) expression. The PG-degrading hydroxyprostaglandin-dehydrogenase-15 (HPGD) expression is only reduced in male cardiomyocytes, which is associated with increased prostaglandin D2 (PGD2) secretion from isolated male but not female CKO-cardiomyocytes. Reduced HPGD expression in male cardiomyocytes derive from impaired androgen receptor (AR)-signaling due to loss of its cofactor STAT3. Elevated PGD2 secretion in males is associated with increased white adipocyte accumulation in aged male but not female hearts. Adipocyte differentiation is enhanced in isolated stem cell antigen-1 (SCA-1)+ cardiac progenitor cells (CPC) from young male CKO-mice compared with the adipocyte differentiation of male wild-type (WT)-CPC and CPC isolated from female mice. Epigenetic analysis in freshly isolated male CKO-CPC display hypermethylation in pro-angiogenic genes (Fgfr2, Epas1) and hypomethylation in the white adipocyte differentiation gene Zfp423 associated with up-regulated ZFP423 expression and a shift from endothelial to white adipocyte differentiation compared with WT-CPC. The expression of the histone-methyltransferase EZH2 is reduced in male CKO-CPC compared with male WT-CPC, whereas no differences in the EZH2 expression in female CPC were observed. Clonally expanded CPC can differentiate into endothelial cells or into adipocytes depending on the differentiation conditions. ZFP423 overexpression is sufficient to induce white adipocyte differentiation of clonal CPC. In isolated WT-CPC, PGD2 stimulation reduces the expression of EZH2, thereby up-regulating ZFP423 expression and promoting white adipocyte differentiation. The treatment of young male CKO mice with the COX inhibitor Ibuprofen or the PGD2 receptor (DP)2 receptor antagonist BAY-u 3405 in vivo increased EZH2 expression and reduced ZFP423 expression and adipocyte differentiation in CKO-CPC. Thus, cardiomyocyte STAT3 deficiency leads to age-related and sex-specific cardiac remodeling and failure in part due to sex-specific alterations in PGD2 secretion and subsequent epigenetic impairment of the differentiation potential of CPC. Causally involved is the impaired AR signaling in absence of STAT3, which reduces the expression of the PG-degrading enzyme HPGD.

Initial WNT/ß-Catenin Activation Enhanced Mesoderm Commitment, Extracellular Matrix Expression, Cell Aggregation and Cartilage Tissue Yield From Induced Pluripotent Stem Cells.

Mesodermal differentiation of induced pluripotent stem cells (iPSCs) in vitro and subsequent specification into mesodermal derivatives like chondrocytes is currently afflicted with a substantial cell loss that severely limits tissue yield. More knowledge on the key players regulating mesodermal differentiation of iPSCs is currently needed to drive all cells into the desired lineage and to overcome the current need for intermediate cell selection steps to remove misdifferentiated cells. Using two independent human iPSC lines, we here report that a short initial WNT/ß-catenin pulse induced by the small molecule CHIR99021 (24 h) enhanced expression of mesodermal markers (PDGFRα, HAND1, KDR, and GATA4), supported the exit from pluripotency (decreased OCT4, SOX2, and LIN28A) and inhibited ectodermal misdifferentiation (reduced PAX6, TUBB3, and NES). Importantly, the initial CHIR pulse increased cell proliferation until day 14 (five-fold), adjusted expression of adhesion-related genes (CDH3 up, CDH6 down) and increased extracellular matrix (ECM)-related gene expression (COL6, COL1, COL3, COL5, DCN, NPNT, LUM, MGP, MATN2, and VTN), thus yielding more matrix-interacting progenitors with a high aggregation capability. Enhanced contribution to chondrogenic pellet formation increased the cell yield after eight weeks 200-fold compared to controls. The collagen type II and proteoglycan-positive area was enlarged in the CHIR group, indicating an increased number of cartilage-forming cells. Conclusively, short initial WNT activation improved mesoderm commitment and our data demonstrated for the first time to our knowledge that, acting via stimulation of cell proliferation, ECM expression and cell aggregation, WNT pulsing is a key step to make cell selection steps before chondrogenesis obsolete. This advanced understanding of the WNT/ß-catenin function is a major step toward robust and efficient generation of high-quality mesodermal progenitors from human iPSCs and toward rescuing low tissue yield during subsequent in vitro chondrogenesis, which is highly desired for clinical cartilage regeneration, disease modeling and drug screening.

Generation of two hiPSC clones (MHHi019-A, MHHi019-B) from a primary ciliary dyskinesia patient carrying a homozygous deletion in the NME5 gene (c.415delA (p.Ile139Tyrfs*8)).

Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by defects in motile cilia and is known to occur in about 1 in 20,000 live births (Horani and Ferkol, 2018). Among the many genes associated with PCD, NME5, a gene encoding a protein involved in ciliary function, was recently reported to be involved in PCD (Anderegg et al., 2019; Cho et al., 2020). We have established two human induced pluripotent stem cell clones from a PCD patient carrying a deletion in the NME5 gene (c.415delA (p.Ile139Tyrfs*8)).

Generation of two hiPSC lines (MHHi016-A, MHHi016-B) from a primary ciliary dyskinesia patient carrying a homozygous 5 bp duplication (c.248_252dup (p.Gly85Cysfs*11)) in exon 1 of the CCNO gene.

Cyclin O (CCNO) is involved in cell cycle regulation and mutations of CCNO are linked to the rare genetic disease primary ciliary dyskinesia (PCD). Mutations in CCNO are associated with reduced cilia number and cilia agenesis on epithelia of the respiratory tract. This article deals with the description of two hiPSC lines generated from a PCD patient carrying a mutation in exon 1 of the CCNO gene. The lines offer a valuable tool for in vitro modeling PCD pathophysiology.

Generation of two human induced pluripotent stem cell lines (MHHi017-A, MHHi017-B) from a patient with primary ciliary dyskinesia carrying a homozygous mutation (c.7915C > T [p.Arg2639*]) in the DNAH5 gene.

Dynein axonemal heavy chain 5 (DNAH5) is part of a microtubule-associated protein complex found within the cilia of the lung. Mutations in the DNAH5 gene lead to impaired ciliary function and are linked to primary ciliary dyskinesia (PCD), a rare autosomal recessive disorder. We established two human induced pluripotent stem cell (hiPSC) lines generated from a patient with PCD and homozygous mutation in the corresponding DNAH5 gene. These cell lines represent an excellent tool for modeling the ciliary dysfunction in PCD.

Generation of an induced pluripotent stem cell line (MHHi018-A) from a patient with Cystic Fibrosis carrying p.Asn1303Lys (N1303K) mutation.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene which encodes for a chloride ion channel regulating the balance of salt and water across secretory epithelia. Here we generated an iPSC line from a CF patient homozygous for the p.Asn1303Lys mutation, a Class II folding defect mutation. This iPSC line provides a useful resource for disease modeling and to investigate the pharmacological response to CFTR modulators in iPSC derived epithelia.

Generation of three induced pluripotent stem cell lines (MHHi012-A, MHHi013-A, MHHi014-A) from a family with Loeys-Dietz syndrome carrying a heterozygous p.M253I (c.759G>A) mutation in the TGFBR1 gene.

Loeys-Dietz syndrome (LDS) is a rare connective tissue disorder characterized by a genetic predisposition for thoracic aortic aneurysm and dissection. Despite heterozygous loss-of-function mutations in genes for ligand, receptor, or downstream mediators of the transforming growth factor ß (TGFß) pathway, LDS is associated with a signature of high TGFß signaling. We generated induced pluripotent stem cell (iPSC) lines from three adult LDS-patients (two male, one female) of a family with a heterozygous point mutation in exon 4 of the TGFß-receptor1 (TGFBR1) gene (p.M253I; c.759G>A). The lines offer a valuable resource for modeling the pathophysiology of genetically mediated aortic disease.