Generation of human induced pluripotential stem cells from individuals with complex heterozygous, isogenic corrected, and homozygous Bloc1s1 mutations.

Genetic studies show that BLOC1S1 modulates mitochondrial and endosome-lysosome function (Wu et al., 2021a). Furthermore, Bloc1s1 mutations are linked to leukodystrophy (Bertoli-Avella et al., 2021). The Vanderver laboratory identified additional individuals with leukodystrophy that harbored either complex heterozygous (Bloc1s1 c.206A > C and c.359G > A), or homozygous (Bloc1s1 c.185 T > C) point mutations. We generated induced pluripotential stem cell (iPSC) lines from these subjects, from parents of the complex heterozygous mutations patient, and from CRISPR isogenic (c.206A > C and c.359G > A) corrected iPSC-line. These complex heterozygous, homozygous, and isogenic-corrected Bloc1s1 lines were phenotypically normal and were capable of differentiation towards the three germ layers.

Sympathetic Neurons Regulate Cardiomyocyte Maturation in Culture.

Embryos devoid of autonomic innervation suffer sudden cardiac death. However, whether autonomic neurons have a role in heart development is poorly understood. To investigate if sympathetic neurons impact cardiomyocyte maturation, we co-cultured phenotypically immature cardiomyocytes derived from human induced pluripotent stem cells with mouse sympathetic ganglion neurons. We found that 1) multiple cardiac structure and ion channel genes related to cardiomyocyte maturation were up-regulated when co-cultured with sympathetic neurons; 2) sarcomere organization and connexin-43 gap junctions increased; 3) calcium imaging showed greater transient amplitudes. However, sarcomere spacing, relaxation time, and level of sarcoplasmic reticulum calcium did not show matured phenotypes. We further found that addition of endothelial and epicardial support cells did not enhance maturation to a greater extent beyond sympathetic neurons, while administration of isoproterenol alone was insufficient to induce changes in gene expression. These results demonstrate that sympathetic neurons have a significant and complex role in regulating cardiomyocyte development.

Four induced pluripotent stem cell lines (TRNDi021-C, TRNDi023-D, TRNDi024-D and TRNDi025-A) generated from fibroblasts of four healthy individuals.

Expanded human skin fibroblast cells from four different aged healthy individuals, 11-year-old female, 1-year-old male, 2-month-old male, and 8-year-old male, were used to generate integration-free induced pluripotent stem cell (iPSC) lines TRNDi021-C, TRNDi023-D, TRNDi024-D, and TRNDi025-A, respectively, by exogenous expression of four reprogramming factors, human SXO2, OCT3/4, C-MYC, KLF4. The authenticity of established iPSC lines was confirmed by the expressions of stem cell markers, karyotype analysis, and teratoma formation. These iPSC lines could serve as young healthy controls for the studies involving patient-specific iPSCs.

Differentiation of Cardiomyocytes from Human Pluripotent Stem Cells in Fully Chemically Defined Conditions.

In the past few years, several different methods for differentiation of directed cardiomyocytes from human pluripotent stem cells (hPSCs) in chemically defined conditions have been reported, including our own (Burridge et al., 2014; Lian et al., 2012; Lin et al., 2017). To help researchers adapt to our simple and cost-effective method, we provide step-by-step protocols for hPSC-cardiomyocyte differentiation, including hPSC culture, cardiomyocyte differentiation, cardiomyocyte passaging, and cryopreservation. For complete details on the use and execution of this protocol, please refer to Lin et al. (2017).

CRISPR/Cas9-mediated introduction of the sodium/iodide symporter gene enables noninvasive in vivo tracking of induced pluripotent stem cell-derived cardiomyocytes.

Techniques that enable longitudinal tracking of cell fate after myocardial delivery are imperative for optimizing the efficacy of cell-based cardiac therapies. However, these approaches have been underutilized in preclinical models and clinical trials, and there is considerable demand for site-specific strategies achieving long-term expression of reporter genes compatible with safe noninvasive imaging. In this study, the rhesus sodium/iodide symporter (NIS) gene was incorporated into rhesus macaque induced pluripotent stem cells (RhiPSCs) via CRISPR/Cas9. Cardiomyocytes derived from NIS-RhiPSCs (NIS-RhiPSC-CMs) exhibited overall similar morphological and electrophysiological characteristics compared to parental control RhiPSC-CMs at baseline and with exposure to physiological levels of sodium iodide. Mice were injected intramyocardially with 2 million NIS-RhiPSC-CMs immediately following myocardial infarction, and serial positron emission tomography/computed tomography was performed with 18 F-tetrafluoroborate to monitor transplanted cells in vivo. NIS-RhiPSC-CMs could be detected until study conclusion at 8 to 10 weeks postinjection. This NIS-based molecular imaging platform, with optimal safety and sensitivity characteristics, is primed for translation into large-animal preclinical models and clinical trials.

Generation of two induced pluripotent stem cell lines (NHLBIi001-A and NHLBIi001-B) from a healthy Caucasian female volunteer with normal cardiac function.

Human-derived induced pluripotent stem cells (iPSCs) have proven to be indispensable in cardiovascular drug development, disease modeling, and developmental biology research. For this reason, it is particularly useful to develop wild-type iPSC lines to be used in experimental or control conditions. Here, we present two such cell lines generated from a sample of peripheral blood mononuclear cells (PBMCs) from a healthy patient with normal cardiac function.

p53 prevents doxorubicin cardiotoxicity independently of its prototypical tumor suppressor activities.

Doxorubicin is a widely used chemotherapeutic agent that causes dose-dependent cardiotoxicity in a subset of treated patients, but the genetic determinants of this susceptibility are poorly understood. Here, we report that a noncanonical tumor suppressor activity of p53 prevents cardiac dysfunction in a mouse model induced by doxorubicin administered in divided low doses as in the clinics. While relatively preserved in wild-type (p53+/+ ) state, mice deficient in p53 (p53-/- ) developed left ventricular (LV) systolic dysfunction after doxorubicin treatment. This functional decline in p53-/- mice was associated with decreases in cardiac oxidative metabolism, mitochondrial mass, and mitochondrial genomic DNA (mtDNA) homeostasis. Notably, mice with homozygous knockin of the p53 R172H (p53172H/H ) mutation, which like p53-/- state lacks the prototypical tumor suppressor activities of p53 such as apoptosis but retains its mitochondrial biogenesis capacity, showed preservation of LV function and mitochondria after doxorubicin treatment. In contrast to p53-null state, wild-type and mutant p53 displayed distinct mechanisms of transactivating mitochondrial transcription factor A (TFAM) and p53-inducible ribonucleotide reductase 2 (p53R2), which are involved in mtDNA transcription and maintenance. Importantly, supplementing mice with a precursor of NAD+ prevented the mtDNA depletion and cardiac dysfunction. These findings suggest that loss of mtDNA contributes to cardiomyopathy pathogenesis induced by doxorubicin administered on a schedule simulating that in the clinics. Given a similar mtDNA protection role of p53 in doxorubicin-treated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, the mitochondrial markers associated with cardiomyopathy development observed in blood and skeletal muscle cells may have prognostic utility.

The ribosomal prolyl-hydroxylase OGFOD1 decreases during cardiac differentiation and modulates translation and splicing.

The mechanisms regulating translation and splicing are not well understood. We provide insight into a new regulator of translation, OGFOD1 (2-oxoglutarate and iron dependent oxygenase domain-containing protein 1), which is a prolyl-hydroxylase that catalyzes the posttranslational hydroxylation of Pro-62 in the small ribosomal protein S23. We show that deletion of OGFOD1 in an in vitro model of human cardiomyocytes decreases translation of specific proteins (e.g., RNA-binding proteins) and alters splicing. RNA sequencing showed poor correlation between changes in mRNA and protein synthesis, suggesting that posttranscriptional regulation was the primary cause for the observed differences. We found that loss of OGFOD1 and the resultant alterations in protein translation modulates the cardiac proteome, shifting it towards higher protein amounts of sarcomeric proteins such as cardiac troponins, titin and cardiac myosin binding protein C. Furthermore, we found a decrease of OGFOD1 during cardiomyocyte differentiation. These results suggest that loss of OGFOD1 modulates protein translation and splicing, thereby leading to alterations in the cardiac proteome and highlight the role of altered translation and splicing in regulating the proteome..

Generation of an induced pluripotent stem cell line (TRNDi003-A) from a Noonan syndrome with multiple lentigines (NSML) patient carrying a p.Q510P mutation in the PTPN11 gene.

Noonan syndrome with multiple lentigines (NSML), formerly known as LEOPARD Syndrome, is a rare autosomal dominant disorder. Approximately 90% of NSML cases are caused by missense mutations in the PTPN11 gene which encodes the protein tyrosine phosphatase SHP2. A human induced pluripotent stem cell (iPSC) line was generated using peripheral blood mononuclear cells (PBMCs) from a patient with NSML that carries a gene mutation of p.Q510P on the PTPN11 gene using non-integrating Sendai virus technique. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development to treat NSML.

Efficient differentiation of cardiomyocytes and generation of calcium-sensor reporter lines from nonhuman primate iPSCs.

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy, but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with >85% purity in 10 days, using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes, we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies, including in vivo tissue regeneration models and drug screening.

Parkin regulation of CHOP modulates susceptibility to cardiac endoplasmic reticulum stress.

The regulatory control of cardiac endoplasmic reticulum (ER) stress is incompletely characterized. As ER stress signaling upregulates the E3-ubiquitin ligase Parkin, we investigated the role of Parkin in cardiac ER stress. Parkin knockout mice exposed to aortic constriction-induced cardiac pressure-overload or in response to systemic tunicamycin (TM) developed adverse ventricular remodeling with excessive levels of the ER regulatory C/EBP homologous protein CHOP. CHOP was identified as a Parkin substrate and its turnover was Parkin-dose and proteasome-dependent. Parkin depletion in cardiac HL-1 cells increased CHOP levels and enhanced susceptibility to TM-induced cell death. Parkin reconstitution rescued this phenotype and the contribution of excess CHOP to this ER stress injury was confirmed by reduction in TM-induced cell death when CHOP was depleted in Parkin knockdown cardiomyocytes. Isogenic Parkin mutant iPSC-derived cardiomyocytes showed exaggerated ER stress induced CHOP and apoptotic signatures and myocardium from subjects with dilated cardiomyopathy showed excessive Parkin and CHOP induction. This study identifies that Parkin functions to blunt excessive CHOP to prevent maladaptive ER stress-induced cell death and adverse cardiac ventricular remodeling. Additionally, Parkin is identified as a novel post-translational regulatory moderator of CHOP stability and uncovers an additional stress-modifying function of this E3-ubiquitin ligase.

Rhesus Macaque iPSC Generation and Maintenance.

The rhesus macaque (Macaca mulatta) is physiologically and phylogenetically similar to humans, and therefore represents an invaluable model for the pre-clinical assessment of the safety and feasibility of iPSC-derived cell therapies. The use of an excisable polycistronic lentiviral STEMCCA vector to reprogram rhesus fibroblasts or bone marrow stromal cells (BMSCs) into RhiPSCs is described. After reprogramming, the pluripotency transgenes can be removed by transient expression of Cre, leaving a residual genetic tag that may be useful for identification of RhiPSC-derived tissues in vivo. Finally, the steps to maintain pluripotency during passaging of RhiPSCs, required for successful utilization of RhiPSCs, is described. © 2017 by John Wiley & Sons, Inc.

Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin-Free Medium, Enabling Consistent Manufacture of Cardiomyocytes.

Cardiomyocytes can be differentiated from human pluripotent stem cells (hPSCs) in defined conditions, but efficient and consistent cardiomyocyte differentiation often requires expensive reagents such as B27 supplement or recombinant albumin. Using a chemically defined albumin-free (E8 basal) medium, we identified heparin as a novel factor that significantly promotes cardiomyocyte differentiation efficiency, and developed an efficient method to differentiate hPSCs into cardiomyocytes. The treatment with heparin helped cardiomyocyte differentiation consistently reach at least 80% purity (up to 95%) from more than 10 different hPSC lines in chemically defined Dulbecco's modified Eagle's medium/F-12-based medium on either Matrigel or defined matrices like vitronectin and Synthemax. One of heparin's main functions was to act as a Wnt modulator that helped promote robust and consistent cardiomyocyte production. Our study provides an efficient, reliable, and cost-effective method for cardiomyocyte derivation from hPSCs that can be used for potential large-scale drug screening, disease modeling, and future cellular therapies. Stem Cells Translational Medicine 2017;6:527-538.

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers.

Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model. A clinically relevant marker gene, human truncated CD19 (hΔCD19), or GFP was inserted into the AAVS1 site in RhiPSCs using the CRISPR/Cas9 system. Genetically modified RhiPSCs maintained normal karyotype and pluripotency, and these clones were able to further differentiate into all three germ layers in vitro and in vivo. In contrast to transgene delivery using randomly integrating viral vectors, AAVS1 targeting allowed stable transgene expression following differentiation. Off-target mutations were observed in some edited clones, highlighting the importance of careful characterization of these cells prior to downstream applications. Genetically marked RhiPSCs will be useful to further advance clinically relevant models for iPSC-based cell therapies.

Snail1-dependent p53 repression regulates expansion and activity of tumour-initiating cells in breast cancer.

The zinc-finger transcription factor Snail1 is inappropriately expressed in breast cancer and associated with poor prognosis. While interrogating human databases, we uncovered marked decreases in relapse-free survival of breast cancer patients expressing high Snail1 levels in tandem with wild-type, but not mutant, p53. Using a Snail1 conditional knockout model of mouse breast cancer that maintains wild-type p53, we find that Snail1 plays an essential role in tumour progression by controlling the expansion and activity of tumour-initiating cells in preneoplastic glands and established tumours, whereas it is not required for normal mammary development. Growth and survival of preneoplastic as well as neoplastic mammary epithelial cells is dependent on the formation of a Snail1/HDAC1/p53 tri-molecular complex that deacetylates active p53, thereby promoting its proteasomal degradation. Our findings identify Snail1 as a molecular bypass that suppresses the anti-proliferative and pro-apoptotic effects exerted by wild-type p53 in breast cancer.

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

AIMS: Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. METHODS AND RESULTS: We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. CONCLUSIONS: This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing.

An Orthologous Epigenetic Gene Expression Signature Derived from Differentiating Embryonic Stem Cells Identifies Regulators of Cardiogenesis.

Here we used predictive gene expression signatures within a multi-species framework to identify the genes that underlie cardiac cell fate decisions in differentiating embryonic stem cells. We show that the overlapping orthologous mouse and human genes are the most accurate candidate cardiogenic genes as these genes identified the most conserved developmental pathways that characterize the cardiac lineage. An RNAi-based screen of the candidate genes in Drosophila uncovered numerous novel cardiogenic genes. shRNA knockdown combined with transcriptome profiling of the newly-identified transcription factors zinc finger protein 503 and zinc finger E-box binding homeobox 2 and the well-known cardiac regulatory factor NK2 homeobox 5 revealed that zinc finger E-box binding homeobox 2 activates terminal differentiation genes required for cardiomyocyte structure and function whereas zinc finger protein 503 and NK2 homeobox 5 are required for specification of the cardiac lineage. We further demonstrated that an essential role of NK2 homeobox 5 and zinc finger protein 503 in specification of the cardiac lineage is the repression of gene expression programs characteristic of alternative cell fates. Collectively, these results show that orthologous gene expression signatures can be used to identify conserved cardiogenic pathways.

Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein.

Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.

A cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells in chemically defined culture.

Factors limiting the adoption of iPSC technology include the cost of developing lines and the time period that it takes to characterize and bank them, particularly when integration free, feeder free, and Xeno-free components are used. In this manuscript we describe our optimization procedure that enables a single technician to make 20-40 lines at a time in a 24-96 well format in a reliable and reproducible fashion. Improvements spanned the entire workflow and included using RNA virus, reducing cytotoxicity of reagents, developing improved transfection and freezing efficiencies, modifying the manual colony picking steps, enhancing passaging efficiency and developing early criteria of success. These modifications allowed us to make more than two hundred well-characterized lines per year.