Role of Cytokines and Growth Factors in the Manufacturing of iPSC-Derived Allogeneic Cell Therapy Products.

Cytokines and other growth factors are essential for cell expansion, health, function, and immune stimulation. Stem cells have the additional reliance on these factors to direct differentiation to the appropriate terminal cell type. Successful manufacturing of allogeneic cell therapies from induced pluripotent stem cells (iPSCs) requires close attention to the selection and control of cytokines and factors used throughout the manufacturing process, as well as after administration to the patient.

Polarized Cytokine Release Triggered by P2X7 Receptor from Retinal Pigmented Epithelial Cells Dependent on Calcium Influx.

Cytokine release from non-inflammatory cells is a key step in innate immunity, and agonists triggering cytokine release are central in coordinating responses. P2X7 receptor (P2X7R) stimulation by extracellular ATP is best known to active the NLRP3 inflammasome and release IL-1β, but stimulation also leads to release of other cytokines. As cytokine signaling by retinal pigmented epithelial (RPE) cells is implicated in retinal neurodegeneration, the role of P2X7R in release of cytokine IL-6 from RPE cells was investigated.

Generation of human control iPSC line CHOPi004-A from juvenile foreskin fibroblast cells.

The CHOPWT4 iPSC line was generated as a control for applications such as differentiation analyses to the three germ layers and derivative tissues. Human foreskin fibroblasts were reprogrammed using the non-integrating Sendai virus expressing Oct3/4, Sox2, c-myc, and Klf4.

Dual SMAD inhibition and Wnt inhibition enable efficient and reproducible differentiations of induced pluripotent stem cells into retinal ganglion cells.

Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo.

A Mini Review: Moving iPSC-Derived Retinal Subtypes Forward for Clinical Applications for Retinal Degenerative Diseases.

Patient-derived human-induced pluripotent stem cells (iPSCs) have been critical in advancing our understanding of the underlying mechanisms of numerous retinal disorders. Many of these retinal disorders have no effective treatment and result in severe visual impairment and even blindness. Among the retinal degenerative diseases modeled by iPSCs are age-related macular degeneration (AMD), glaucoma, Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), and autosomal dominant retinitis pigmentosa (adRP).

CRISPR Activation Enhances Potency of AAV Vectors Driven by Tissue-Specific Promoters.

Validation of gene transfer vectors containing tissue-specific promoters in cell-based functional assays poses a formidable challenge for gene therapy product development. Here, we describe a novel approach based on CRISPR/dCas9 transcriptional activation to achieve robust transgene expression from transgene cassettes containing tissue or cell type-specific promoters after infection with AAV vectors in cell-based systems. Guide RNA sequences targeting two promoters that are highly active within mammalian photoreceptors were screened in a novel promoter activation assay.

Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1-9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons.

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons.

Functional Cortical Axon Tracts Generated from Human Stem Cell-Derived Neurons.

Axon regeneration is negligible in the adult mammalian brain, and thus, white matter damage often leads to permanent neurological deficits. A novel approach for axon repair is the generation of axon tracts in the laboratory setting followed by transplantation of these constructs. This article details a human substrate for this repair strategy.

Use of induced pluripotent stem cell models to probe the pathogenesis of Choroideremia and to develop a potential treatment.

Choroideremia (CHM) is a rare monogenic, X-linked recessive inherited retinal degeneration resulting from mutations in the Rab Escort Protein-1 (REP1) encoding CHM gene. The primary retinal cell type leading to CHM is unknown. In this study, we explored the utility of induced pluripotent stem cell-derived models of retinal pigmented epithelium (iPSC-RPE) to study disease pathogenesis and a potential gene-based intervention in four different genetically distinct forms of CHM.

NIPBL haploinsufficiency reveals a constellation of transcriptome disruptions in the pluripotent and cardiac states.

Cornelia de Lange syndrome (CdLS) is a complex disorder with multiple structural and developmental defects caused by mutations in structural and regulatory proteins involved in the cohesin complex. NIPBL, a cohesin regulatory protein, has been identified as a critical protein responsible for the orchestration of transcriptomic regulatory networks necessary for embryonic development. Mutations in NIPBL are responsible for the majority of cases of CdLS.

Strategies for retinal cell generation from human pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are specialized self-renewing cells that are generated by exogenously expressing pluripotency-associated transcription factors in somatic cells such as fibroblasts, peripheral blood mononuclear cells, or lymphoblastoid cell lines (LCLs). iPSCs are functionally similar to naturally pluripotent embryonic stem cells (ESCs) in their capacity to propagate indefinitely and potential to differentiate into all human cell types, and are devoid of the associated ethical complications of origin. iPSCs are useful for studying embryonic development, disease modeling, and drug screening.

Cornelia de Lange syndrome and molecular implications of the cohesin complex: Abstracts from the 7th biennial scientific and educational symposium 2016.

Cornelia de Lange Syndrome (CdLS) is due to mutations in the genes for the structural and regulatory proteins that make up the cohesin complex, and is considered a cohesinopathy disorder or, more recently, a transcriptomopathy. New phenotypes have been recognized in this expanding field. There are multiple clinical issues facing individuals with all forms of CdLS, particularly in the neurodevelopmental system, but also gastrointestinal, cardiac, and musculoskeletal.

Generation of Hermansky Pudlak syndrome type 2 (HPS2) induced pluripotent stem cells (iPSCs).

Hermansky-Pudlak syndrome type 2 (HPS2) is a rare autosomal recessive disorder resulting from functional mutations in the adaptor-related protein complex 3, beta 1 subunit (AP3B1) gene. This gene plays a role in organelle biogenesis associated with melanosomes, platelet dense granules, and lysosomes. Here we describe the generation of an HPS2 iPS cell line (CHOPHPS2) using a Cre-excisable polycistronic STEMCCA lentivirus.

Generation of Hermansky-Pudlak Syndrome Type 1 (HPS1) induced pluripotent stem cells (iPSCs).

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by deficiencies in lysosome-related organelles such as melanosomes and platelet-dense granules. The disorder is classified into nine different subtypes (HPS1-HPS9) based on genetic mutations in 9 unique genes. Here we describe the generation of an HPS1 iPSC line (CHOPHPS1) using a Cre-excisable polycistronic STEMCCA lentivirus.

Generation of human control iPS cell line CHOPWT9 from healthy adult peripheral blood mononuclear cells.

The CHOPWT9 induced pluripotent stem (iPS) cell line was generated for use as a control for applications such as differentiation analyses to the three germ layers and derivative tissues. Peripheral blood mononuclear cells (PBMCs) obtained from a healthy adult female were reprogrammed using non-integrating Sendai viral vectors expressing Oct3/4, Sox2, c-Myc, and Klf4.

Retinas in a Dish Peek into Inherited Retinal Degeneration.

Human retinal degeneration can cause blindness, and the lack of relevant model systems has made identifying underlying mechanisms challenging. Parfitt et al. (2016) generate three-dimensional retinal tissue from patient-derived induced pluripotent stem cells to identify how CEP290 mutations cause retinal degeneration, and show an antisense approach can correct disease-associated phenotypes.

Generation of poikiloderma with neutropenia (PN) induced pluripotent stem cells (iPSCs).

Poikiloderma with neutropenia (PN, Clericuzio-type poikiloderma with neutropenia) is a rare autosomal recessive disorder caused by biallelic mutations in the USB1 gene (Alias C16orf57 and MPN1). To date, there have been only 37 reported cases worldwide of this disorder that presents with neutropenia, early onset poikiloderma, respiratory infections, palmo-plantar hyperkeratosis, and skeletal defects. Here we described the generation of human induced pluripotent stem cell lines (PN1 and PN2) from the peripheral blood of a 1-year-old patient using the dox-inducible STEMCCA vector.

OCT4 Coordinates with WNT Signaling to Pre-pattern Chromatin at the SOX17 Locus during Human ES Cell Differentiation into Definitive Endoderm.

We demonstrate that the pluripotency gene OCT4 has a role in regulating differentiation via Wnt signaling. OCT4 expression levels in human embryonic stem cells increases transiently during the first 24 hr of in vitro differentiation, with OCT4 occupancy increasing at endoderm regulators such as SOX17 and FOXA2. This increased occupancy correlates with loss of the PRC2 complex and the inhibitory histone mark H3K27me3.

Dysregulation of the Transforming Growth Factor β Pathway in Induced Pluripotent Stem Cells Generated from Patients with Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome with clinical features of red cell aplasia and variable developmental abnormalities. Most affected patients have heterozygous loss of function mutations in ribosomal protein genes but the pathogenic mechanism is still unknown. We generated induced pluripotent stem cells from DBA patients carrying RPS19 or RPL5 mutations.

Clinical, developmental and molecular update on Cornelia de Lange syndrome and the cohesin complex: abstracts from the 2014 Scientific and Educational Symposium.

Cornelia de Lange Syndrome (CdLS) is the most common example of disorders of the cohesin complex, or cohesinopathies. There are a myriad of clinical issues facing individuals with CdLS, particularly in the neurodevelopmental system, which also have implications for the parents and caretakers, involved professionals, therapists, and schools. Basic research in developmental and cell biology on cohesin is showing significant progress, with improved understanding of the mechanisms and the possibility of potential therapeutics.

Emergence of a stage-dependent human liver disease signature with directed differentiation of alpha-1 antitrypsin-deficient iPS cells.

Induced pluripotent stem cells (iPSCs) provide an inexhaustible source of cells for modeling disease and testing drugs. Here we develop a bioinformatic approach to detect differences between the genomic programs of iPSCs derived from diseased versus normal human cohorts as they emerge during in vitro directed differentiation. Using iPSCs generated from a cohort carrying mutations (PiZZ) in the gene responsible for alpha-1 antitrypsin (AAT) deficiency, we find that the global transcriptomes of PiZZ iPSCs diverge from normal controls upon differentiation to hepatic cells.

Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia.

To explore how RUNX1 mutations predispose to leukemia, we generated induced pluripotent stem cells (iPSCs) from 2 pedigrees with germline RUNX1 mutations. The first, carrying a missense R174Q mutation, which acts as a dominant-negative mutant, is associated with thrombocytopenia and leukemia, and the second, carrying a monoallelic gene deletion inducing a haploinsufficiency, presents only as thrombocytopenia. Hematopoietic differentiation of these iPSC clones demonstrated profound defects in erythropoiesis and megakaryopoiesis and deregulated expression of RUNX1 targets.

Efficient iPS cell generation from blood using episomes and HDAC inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming.