The evaluation expression of non-coding RNAs in response to HSV-G47∆ oncolytic virus infection in glioblastoma multiforme cancer stem cells.

Glioblastoma multiforme is the most aggressive astrocytes brain tumor. Glioblastoma cancer stem cells and hypoxia conditions are well-known major obstacles in treatment. Studies have revealed that non-coding RNAs serve a critical role in glioblastoma progression, invasion, and resistance to chemo-radiotherapy. The present study examined the expression levels of microRNAs (in normoxic condition) and long non-coding RNAs (in normoxic and hypoxic conditions) in glioblastoma stem cells treated with the HSV-G47∆. The expression levels of 43 miRNAs and 8 lncRNAs isolated from U251-GBM-CSCs were analyzed using a miRCURY LNA custom PCR array and a quantitative PCR assay, respectively. The data revealed that out of 43 miRNAs that only were checked in normoxic condition, the only 8 miRNAs, including miR-7-1, miR-let-7b, miR-130a, miR-137, miR-200b, miR-221, miR-222, and miR-874, were markedly upregulated. The expression levels of lncRNAs, including LEF1 antisense RNA 1 (LEF1-AS1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), long intergenic non-protein coding RNA 470 (LINC00470), tumor suppressor candidate 7 (TUSC7), HOX transcript antisense RNA (HOTAIR), nuclear paraspeckle assembly transcript 1 (NEAT1), and X inactive specific transcript (XIST), were markedly downregulated in the hypoxic microenvironment, and H19-imprinted maternally expressed transcript (H19) was not observed to be dysregulated in this environment. Under normoxic conditions, LEF1-AS1, MALAT1, LINC00470, H19, HOTAIR, NEAT1, and XIST were downregulated and TUSC7 was not targeted by HSV-G47∆. Overall, the present data shows HSVG47Δ treatment deregulates non-coding RNA expression in GBM-CSC tumor microenvironments.

Computational fluid dynamics modeling, a novel, and effective approach for developing scalable cell therapy manufacturing processes.

Induced pluripotent stem cells (iPSCs) hold great potential to generate novel, curative cell therapy products. However, current methods to generate these novel therapies lack scalability, are labor-intensive, require a large footprint, and are not suited to meet clinical and commercial demands. Therefore, it is necessary to develop scalable manufacturing processes to accommodate the generation of high-quality iPSC derivatives under controlled conditions. The current scale-up methods used in cell therapy processes are based on empirical, geometry-dependent methods that do not accurately represent the hydrodynamics of 3D bioreactors. These methods require multiple iterations of scale-up studies, resulting in increased development cost and time. Here we show a novel approach using computational fluid dynamics modeling to effectively scale-up cell therapy manufacturing processes in 3D bioreactors. Using a GMP-compatible iPSC line, we translated and scaled-up a small-scale cardiomyocyte differentiation process to a 3-L computer-controlled bioreactor in an efficient manner, showing comparability in both systems.

Long-Term Stability and Differentiation Potential of Cryopreserved cGMP-Compliant Human Induced Pluripotent Stem Cells.

The clinical effectiveness of human induced pluripotent stem cells (iPSCs) is highly dependent on a few key quality characteristics including the generation of high quality cell bank, long-term genomic stability, post-thaw viability, plating efficiency, retention of pluripotency, directed differentiation, purity, potency, and sterility. We have already reported the establishment of iPSC master cell banks (MCBs) and working cell banks (WCBs) under current good manufacturing procedure (cGMP)-compliant conditions. In this study, we assessed the cellular and genomic stability of the iPSC lines generated and cryopreserved five years ago under cGMP-compliant conditions. iPSC lines were thawed, characterized, and directly differentiated into cells from three germ layers including cardiomyocytes (CMs), neural stem cells (NSCs), and definitive endoderm (DE). The cells were also expanded in 2D and 3D spinner flasks to evaluate their long-term expansion potential in matrix-dependent and feeder-free culture environment. All three lines successfully thawed and attached to the L7TM matrix, and formed typical iPSC colonies that expressed pluripotency markers over 15 passages. iPSCs maintained their differentiation potential as demonstrated with spontaneous and directed differentiation to the three germ layers and corresponding expression of specific markers, respectfully. Furthermore, post-thaw cells showed normal karyotype, negative mycoplasma, and sterility testing. These cells maintained both their 2D and 3D proliferation potential after five years of cryopreservation without acquiring karyotype abnormality, loss of pluripotency, and telomerase activity. These results illustrate the long-term stability of cGMP iPSC lines, which is an important step in establishing a reliable, long-term source of starting materials for clinical and commercial manufacturing of iPSC-derived cell therapy products.

Human induced pluripotent stem cell-derived lung progenitor and alveolar epithelial cells attenuate hyperoxia-induced lung injury.

BACKGROUND AIMS: Bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by disrupted lung growth, is the most common complication in extreme premature infants. BPD leads to persistent pulmonary disease later in life. Alveolar epithelial type 2 cells (AEC2s), a subset of which represent distal lung progenitor cells (LPCs), promote normal lung growth and repair. AEC2 depletion may contribute to persistent lung injury in BPD. We hypothesized that induced pluripotent stem cell (iPSC)-derived AECs prevent lung damage in experimental oxygen-induced BPD. METHODS: Mouse AECs (mAECs), miPSCs/mouse embryonic stem sells, human umbilical cord mesenchymal stromal cells (hUCMSCs), human (h)iPSCs, hiPSC-derived LPCs and hiPSC-derived AECs were delivered intratracheally to hyperoxia-exposed newborn mice. Cells were pre-labeled with a red fluorescent dye for in vivo tracking. RESULTS: Airway delivery of primary mAECs and undifferentiated murine pluripotent cells prevented hyperoxia-induced impairment in lung function and alveolar growth in neonatal mice. Similar to hUCMSC therapy, undifferentiated hiPSCs also preserved lung function and alveolar growth in hyperoxia-exposed neonatal NOD/SCID mice. Long-term assessment of hiPSC administration revealed local teratoma formation and cellular infiltration in various organs. To develop a clinically relevant cell therapy, we used a highly efficient method to differentiate hiPSCs into a homogenous population of AEC2s. Airway delivery of hiPSC-derived AEC2s and hiPSC-derived LPCs, improved lung function and structure and resulted in long-term engraftment without evidence of tumor formation. CONCLUSIONS: hiPSC-derived AEC2 therapy appears effective and safe in this model and warrants further exploration as a therapeutic option for BPD and other lung diseases characterized by AEC injury.

Derivation of iPSCs in stirred suspension bioreactors.

Induced pluripotent stem cells (iPSCs) are typically derived in adherent culture. Here we report fast and efficient derivation of mouse iPSCs in stirred suspension bioreactors, with and without the use of c-Myc. Suspension-reprogrammed cells expressed pluripotency markers, showed multilineage differentiation in vitro and in vivo, and contributed to the germline in chimeric mice. Suspension reprogramming has the potential to accelerate and standardize iPSC research.

Characterization of human induced pluripotent stems cells: Current approaches, challenges, and future solutions.

Human induced pluripotent stem cells (iPSC) have demonstrated massive potentials for use in regenerative and personalized medicine due to their ability to expand in culture and differentiate into specialized cells with therapeutic benefits. However, in order to industrialize iPSC-derived therapies, it is necessary to address the existing challenges surrounding the analytics implemented in the manufacturing process to evaluate and monitor cell expansion, differentiation, and quality of the final products. Here, we review some of the key analytical methods used as part of identity, potency, or safety for in-process or final product release testing and highlighted the challenges and potential solutions for consideration in the Chemistry, Manufacturing and Controls (CMC) strategy for iPSC-based therapies. Some of the challenges associated with characterization and testing of iPSC-based products are related to the choice of analytical technology (to ensure fit-for-purpose), assay reliability and robustness. Automation of analytical methods may be required to reduce hands on time, and improve reliability of the methods through reducing assay variability. Indeed, we have shown that automation of analytical methods is feasible (evaluated using an ELISA based assay) and would result in more precise measurements (demonstrated by lower co-efficient of Variation and standard deviation), less hands-on time, and swift compared to a manually run assay. Therefore, in order to support commercialization of iPSC-based therapies we suggest a well-designed testing strategy to be established in the development phase while incorporating robust, reproducible, reliable, and potentially automated analytics in the manufacturing process.

Returning to the stem state: epigenetics of recapitulating pre-differentiation chromatin structure.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can self-renew indefinitely and contribute to all tissue types of the adult organism. Stem cell-based therapeutic approaches hold enormous promise for the cure of regenerative diseases. Over the last few years, several studies have attempted to decipher the important role of transcription factor networks and epigenetic regulatory signals in the maintenance of ESC pluripotency, but the exact underlying mechanisms have yet to be identified. Among the epigenetic factors, chromatin dynamics and structure have been found to contribute greatly to maintenance of pluripotency and regulation of differentiation in ESCs. These modifications include: covalent histone acetylation and methylation, histone bivalents and chromatin remodeling, and DNA methylation. Studies in ESCs have shown that genes associated with early development are arranged within a bivalent chromatin structure. This is thought to be a "poised yet repressed" situation, which can be activated upon differentiation. The breakthrough of iPSCs has opened a new era in stem cell biology. During reprogramming, the chromatin state of differentiated cells is reset to an embryonic form via a largely unknown mechanism. In this review, the fundamental impact of chromatin dynamic in ESCs as well as its critical role in the generation of iPSCs is discussed.

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes.

BACKGROUND: Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. RESULTS: Murine D3-MHC-neo(r) ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. CONCLUSIONS: This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells.

Addressing Manufacturing Challenges for Commercialization of iPSC-Based Therapies.

The development of reprogramming technology to generate human induced pluripotent stem cells (iPSCs) has tremendously influenced the field of regenerative medicine and clinical therapeutics where curative cell replacement therapies can be used in the treatment of devastating diseases such as Parkinson's disease (PD) and diabetes. In order to commercialize these therapies to treat a large number of individuals, it is important to demonstrate the safety and efficacy of these therapies and ensure that the manufacturing process for iPSC-derived functional cells can be industrialized at an affordable cost. However, there are a number of manufacturing obstacles that need to be addressed in order to meet this vision. It is important to note that the manufacturing process for generation of iPSC-derived specialized cells is relatively long and fairly complex and requires differentiation of high-quality iPSCs into specialized cells in a controlled manner. In this chapter, we have summarized our efforts to address the main challenges present in the industrialization of iPSC-derived cell therapy products with focus on the development of a current Good Manufacturing Practice (cGMP)-compliant iPSC manufacturing process, a comprehensive iPSC characterization platform, long-term stability of cGMP compliant iPSCs, and innovative technologies to address some of the scale-up challenges in establishment of iPSC processing in 3D computer-controlled bioreactors.

High rate of mutation in mitochondrial DNA displacement loop region in human colorectal cancer.

PURPOSE: Mitochondrial DNA mutations are found in many kinds of human cancer and the 1.1 kb displacement loop region has been identified as a "hot spot" for mutation in mitochondrial DNA of tumors. This study evaluated the mutation frequencies in hypervariable regions of mitochondrial displacement loop in patients with colorectal cancer. METHODS: We examined the frequency of mutations in the mitochondrial DNA displacement loop region of 40 colorectal cancer samples in comparison to 150 samples from people without any type of familial cancer history, by automated DNA sequencing. Alignment was made with the revised Cambridge Reference Sequence and any differences recorded as single base substitution, insertions, and deletions. RESULTS: Our results showed that the rate of displacement loop variations was higher in colorectal cancer patients than controls. Nineteen single nucleotide polymorphisms were found; among them eighteen occurred in the displacement loop region. CONCLUSIONS: Mutations in mtDNA D-loop region probably do not cause colorectal cancer but are more likely to be epiphenomena; patients with the high mtDNA variants are at a higher risk of colorectal cancer.

Human-Induced Pluripotent Stem Cells Manufactured Using a Current Good Manufacturing Practice-Compliant Process Differentiate Into Clinically Relevant Cells From Three Germ Layers.

The discovery of reprogramming and generation of human-induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine and opened new opportunities in cell replacement therapies. While generation of iPSCs represents a significant breakthrough, the clinical relevance of iPSCs for cell-based therapies requires generation of high-quality specialized cells through robust and reproducible directed differentiation protocols. We have recently reported manufacturing of human iPSC master cell banks (MCB) under current good manufacturing practices (cGMPs). Here, we describe the clinical potential of human iPSCs generated using this cGMP-compliant process by differentiating them into the cells from all three embryonic germ layers including ectoderm, endoderm, and mesoderm. Most importantly, we have shown that our iPSC manufacturing process and cell culture system is not biased toward a specific lineage. Following controlled induction into a specific differentiation lineage, specialized cells with morphological and cellular characteristics of neural stem cells, definitive endoderm, and cardiomyocytes were developed. We believe that these cGMP-compliant iPSCs have the potential to make various clinically relevant products suitable for cell therapy applications.

Complex I deficiency in Persian multiple sclerosis patients.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system characterized by the morphological hallmarks of inflammation, demyelination and axonal loss. Until now, little attention has been paid to the contribution of mitochondrial respiratory chain enzyme activities to MS. In this study, kinetic analysis of mitochondrial respiratory chain complex I enzyme (measured as NADH-ferricyanide reductase) was performed on intact mitochondria isolated from fresh skeletal muscle in MS patients (n = 10) and control subjects (n = 11). Mitochondrial DNA common deletion and deletions were also tested in MS patients. Our findings showed that complex I activities were significantly reduced (P = 0.007) in patients compared with control. However, we could not find deletion in mtDNA of patients with MS. The presupposition of relationship between MS and mitochondrial disorders is due to predominant maternal transmission of MS in affected parent-child pairs, pathoaetiological role of respiratory chain dysfunction in multisystem disorders and important role of it in neurodegenerative disorders, a number of patients such as LHON or other mtDNA abnormality with developed neurological symptoms indistinguishable from MS and similarity of clinical symptoms in mitochondrial disorders to those of MS. This study suggested that a biochemical defect in complex I activity may be involved in pathogenesis of MS.

Expansion and long-term maintenance of induced pluripotent stem cells in stirred suspension bioreactors.

Induced pluripotent stem cells (iPSCs) can provide an important source of cells for the next-generation of cell therapies in regenerative medicine, in part due to their similarity to embryonic stem cells (ESCs). Patient-specific iPSCs represent an opportunity for autologous cell therapies that are not restricted by immunological, ethical and technical obstacles. One of the technical hurdles that must be overcome before iPSCs can be clinically implemented is the scalable, reproducible production of iPSCs and their differentiated progeny. All of the iPSC lines established thus far have been generated and expanded with static tissue culture protocols, which are time-consuming and suffer from batch-to-batch variability. Alternatively, stirred suspension bioreactors propose several benefits and their homogeneous culture environment facilitates the large-scale expansion required for clinical studies at less cost. We have previously developed protocols for expanding murine and human ESCs as undifferentiated aggregates in stirred suspension bioreactors. The resulting cells were karyotypically normal, expressed pluripotency markers and could be differentiated into all three germ lineages, both in vitro and in vivo. In this study, we demonstrate that stirred suspension bioreactors yield 58-fold expansion of undifferentiated pluripotent iPSCs over 4 days. In vitro differentiation into cartilage, bone and cardiomyocytes lineages, in addition to in vivo teratoma formation, further confirmed the existence of fully functional and undifferentiated pluripotent iPSC aggregates following long-term passaging. Stirred suspension bioreactor culture represents an efficient process for the large-scale expansion and maintenance of iPSCs, which is an important first step in their clinical application.