Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule.

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor ß (TGF-ß) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in vivo and ex vivo models of ocular and lung fibrosis.

Correction to: Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

The original article [1] contains an error in the legend of Fig 5 whereby the descriptions for panels 5d and 5e are incorrect; as such, the corrected legend can be viewed below with its respective figure images.

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

BACKGROUND: Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. METHODS: We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. RESULTS: Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. CONCLUSIONS: This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.

Development of a Three-Dimensional Bioengineering Technology to Generate Lung Tissue for Personalized Disease Modeling.

Stem cell technologies, especially patient-specific, induced stem cell pluripotency and directed differentiation, hold great promise for changing the landscape of medical therapies. Proper exploitation of these methods may lead to personalized organ transplants, but to regenerate organs, it is necessary to develop methods for assembling differentiated cells into functional, organ-level tissues. The generation of three-dimensional human tissue models also holds potential for medical advances in disease modeling, as full organ functionality may not be necessary to recapitulate disease pathophysiology. This is specifically true of lung diseases where animal models often do not recapitulate human disease. Here, we present a method for the generation of self-assembled human lung tissue and its potential for disease modeling and drug discovery for lung diseases characterized by progressive and irreversible scarring such as idiopathic pulmonary fibrosis (IPF). Tissue formation occurs because of the overlapping processes of cellular adhesion to multiple alveolar sac templates, bioreactor rotation, and cellular contraction. Addition of transforming growth factor-ß1 to single cell-type mesenchymal organoids resulted in morphologic scarring typical of that seen in IPF but not in two-dimensional IPF fibroblast cultures. Furthermore, this lung organoid may be modified to contain multiple lung cell types assembled into the correct anatomical location, thereby allowing cell-cell contact and recapitulating the lung microenvironment. Our bottom-up approach for synthesizing patient-specific lung tissue in a scalable system allows for the development of relevant human lung disease models with the potential for high throughput drug screening to identify targeted therapies. Stem Cells Translational Medicine 2017;6:622-633.

High-throughput physical phenotyping of cell differentiation.

In this report, we present multiparameter deformability cytometry (m-DC), in which we explore a large set of parameters describing the physical phenotypes of pluripotent cells and their derivatives. m-DC utilizes microfluidic inertial focusing and hydrodynamic stretching of single cells in conjunction with high-speed video recording to realize high-throughput characterization of over 20 different cell motion and morphology-derived parameters. Parameters extracted from videos include size, deformability, deformation kinetics, and morphology. We train support vector machines that provide evidence that these additional physical measurements improve classification of induced pluripotent stem cells, mesenchymal stem cells, neural stem cells, and their derivatives compared to size and deformability alone. In addition, we utilize visual interactive stochastic neighbor embedding to visually map the high-dimensional physical phenotypic spaces occupied by these stem cells and their progeny and the pathways traversed during differentiation. This report demonstrates the potential of m-DC for improving understanding of physical differences that arise as cells differentiate and identifying cell subpopulations in a label-free manner. Ultimately, such approaches could broaden our understanding of subtle changes in cell phenotypes and their roles in human biology.

A Single CRISPR-Cas9 Deletion Strategy that Targets the Majority of DMD Patients Restores Dystrophin Function in hiPSC-Derived Muscle Cells.

Mutations in DMD disrupt the reading frame, prevent dystrophin translation, and cause Duchenne muscular dystrophy (DMD). Here we describe a CRISPR/Cas9 platform applicable to 60% of DMD patient mutations. We applied the platform to DMD-derived hiPSCs where successful deletion and non-homologous end joining of up to 725 kb reframed the DMD gene. This is the largest CRISPR/Cas9-mediated deletion shown to date in DMD. Use of hiPSCs allowed evaluation of dystrophin in disease-relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from reframed hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo. Furthermore, miR31 was reduced upon reframing, similar to observations in Becker muscular dystrophy. This work demonstrates the feasibility of using a single CRISPR pair to correct the reading frame for the majority of DMD patients.

Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions.

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status.

INTRODUCTION: The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. METHODS: In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. RESULTS: We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. CONCLUSION: For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.

From skin biopsy to neurons through a pluripotent intermediate under Good Manufacturing Practice protocols.

The clinical application of human-induced pluripotent stem cells (hiPSCs) requires not only the production of Good Manufacturing Practice-grade (GMP-grade) hiPSCs but also the derivation of specified cell types for transplantation under GMP conditions. Previous reports have suggested that hiPSCs can be produced in the absence of animal-derived reagents (xenobiotics) to ease the transition to production under GMP standards. However, to facilitate the use of hiPSCs in cell-based therapeutics, their progeny should be produced not only in the absence of xenobiotics but also under GMP conditions requiring extensive standardization of protocols, documentation, and reproducibility of methods and product. Here, we present a successful framework to produce GMP-grade derivatives of hiPSCs that are free of xenobiotic exposure from the collection of patient fibroblasts, through reprogramming, maintenance of hiPSCs, identification of reprogramming vector integration sites (nrLAM-PCR), and finally specification and terminal differentiation of clinically relevant cells. Furthermore, we developed a primary set of Standard Operating Procedures for the GMP-grade derivation and differentiation of these cells as a resource to facilitate widespread adoption of these practices.

Induction of Nrf2 and xCT are involved in the action of the neuroprotective antibiotic ceftriaxone in vitro.

In amyotrophic lateral sclerosis, down-regulation of the astrocyte-specific glutamate excitatory amino acid transporter 2 is hypothesized to increase extracellular glutamate, thereby leading to excitotoxic motor neuron death. The antibiotic ceftriaxone was recently reported to induce excitatory amino acid transporter 2 and to prolong the survival of mutant superoxide dismutase 1 transgenic mice. Here we show that ceftriaxone also protects fibroblasts and the hippocampal cell line HT22, which are not sensitive to excitotoxicity, against oxidative glutamate toxicity, where extracellular glutamate blocks cystine import via the glutamate/cystine-antiporter system x(c)(-). Lack of intracellular cystine leads to glutathione depletion and cell death because of oxidative stress. Ceftriaxone increased system x(c)(-) and glutathione levels independently of its effect on excitatory amino acid transporters by induction of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), a known inducer of system x(c)(-), and the specific x(c)(-) subunit xCT. No significant effect was apparent in fibroblasts deficient in Nrf2 or xCT. Similar ceftriaxone-stimulated changes in Nrf2, system x(c)(-), and glutathione were observed in rat cortical and spinal astrocytes. In addition, ceftriaxone induced xCT mRNA expression in stem cell-derived human motor neurons. We conclude that ceftriaxone-mediated neuroprotection might relate more strongly to activation of the antioxidant defense system including Nrf2 and system x(c)(-) than to excitatory amino acid transporter induction.

Directed differentiation of human-induced pluripotent stem cells generates active motor neurons.

The potential for directed differentiation of human-induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS-derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS-derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS-derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases.

Human embryonic stem cell-derived motor neurons expressing SOD1 mutants exhibit typical signs of motor neuron degeneration linked to ALS.

Human embryonic stem cell (hESC)-derived neurons have the potential to model neurodegenerative disorders. Here, we demonstrate the expression of a mutant gene, superoxide dismutase 1(SOD1), linked to familial amyotrophic lateral sclerosis (ALS) in hESC-derived motor neurons. Green fluorescent protein (GFP) expression under the control of the HB9 enhancer was used to identify SOD1-transfected motor neurons that express human wild-type SOD1 or one of three different mutants (G93A, A4V and I113T) of SOD1. Neurons transfected with mutant SOD1 exhibited reduced cell survival and shortened axonal processes as compared with control-transfected cells, which could survive for 3 weeks or more. The results indicate that hESC-derived cell populations can be directed to express disease-relevant genes and to display characteristics of the disease-specific cell type. These genetically manipulated hESC-derived motor neurons can facilitate and advance the study of disease-specific cellular pathways, and serve as a model system to test new therapeutic approaches.